RESUMEN
Objective: The aim of this study was to determine the plasma pharmacokinetics of oxytetracycline (OTC) in rainbow trout (Oncorhynchus mykiss) of different body sizes. Methods: The research was carried out on three groups as small (30-50 g), medium (90-110 g) and large (185-215 g) body sizes at 8 ± 0.5 °C. OTC was administered orally at a dose of 60 mg/kg to all groups. Blood samples were taken at 19 different sampling times until the 384 h after oxytetracycline administration. The plasma concentrations of OTC were measured using high pressure liquid chromatography-ultraviolet and pharmacokinetic parameters were evaluated using non-compartmental analysis. Results: OTC was detected in small-body sized fish until the 336 h and in medium and large-body sized fish until the 384 h. The elimination half-life of OTC was 85.46, 87.24 and 86.98 h in the small, medium and large body size groups, respectively. The peak plasma concentration increased from 0.66 to 1.11 µg/mL, and the area under the plasma concentration-versus time curve from zero (0) h to infinity (∞) increased from 87.86 to 151.52 h*µg/mL, in tandem with the increase in fish body size. As fish body size increased, volume of distribution and total body clearance decreased. Conclusion: These results show that the pharmacokinetics of OTC vary depending on fish size. Therefore, there is a need to reveal the pharmacodynamic activity of OTC in rainbow trout of different body sizes.
Asunto(s)
Antibacterianos , Tamaño Corporal , Oncorhynchus mykiss , Oxitetraciclina , Animales , Oncorhynchus mykiss/metabolismo , Oxitetraciclina/farmacocinética , Oxitetraciclina/sangre , Oxitetraciclina/administración & dosificación , Administración Oral , Antibacterianos/farmacocinética , Antibacterianos/administración & dosificación , Antibacterianos/sangre , Semivida , Cromatografía Líquida de Alta PresiónRESUMEN
Systemic inflammation elicits sickness behaviors and fever by engaging a complex neuronal circuitry that begins in the preoptic area of the hypothalamus. Ectotherms such as teleost fish display sickness behaviors in response to infection or inflammation, seeking warmer temperatures to enhance survival via behavioral fever responses. To date, the hypothalamus is the only brain region implicated in sickness behaviors and behavioral fever in teleosts. Yet, the complexity of neurobehavioral manifestations underlying sickness responses in teleosts suggests engagement of higher processing areas of the brain. Using in vivo models of systemic inflammation in rainbow trout, we find canonical pyrogenic cytokine responses in the hypothalamus whereas in the telencephalon and the optic tectum il-1b and tnfa expression is decoupled from il-6 expression. Polyamine metabolism changes, characterized by accumulation of putrescine and decreases in spermine and spermidine, are recorded in the telencephalon but not hypothalamus upon systemic injection of bacteria. While systemic inflammation causes canonical behavioral fever in trout, blockade of bacterial polyamine metabolism prior to injection abrogates behavioral fever, polyamine responses, and telencephalic but not hypothalamic cytokine responses. Combined, our work identifies the telencephalon as a neuronal substrate for brain responses to systemic inflammation in teleosts and uncovers the role of polyamines as critical chemical mediators in sickness behaviors.
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Inflamación , Oncorhynchus mykiss , Poliaminas , Telencéfalo , Animales , Telencéfalo/metabolismo , Poliaminas/metabolismo , Inflamación/metabolismo , Oncorhynchus mykiss/metabolismo , Oncorhynchus mykiss/inmunología , Neuronas/metabolismo , Hipotálamo/metabolismo , Espermina/metabolismo , Putrescina/metabolismo , Conducta de Enfermedad/fisiología , Espermidina/metabolismoRESUMEN
Identifying pathogenic microorganisms causing disease is important for epidemiological research, antimicrobial therapy, and control. The current study was carried out to use different methods for the identification of Vibrio anguillarum from diseased rainbow trout (Oncorhynchus mykiss) obtained from Türkiye (Mugla-Fethiye), the damage caused by the pathogenic microorganism in the tissues and organs, and the determination of the antibiotic effective against the pathogen. Hemorrhagic and ulcerative skin lesions and diffuse petechial hemorrhage in the internal organs were clinically detected in diseased fish obtained from the rainbow trout farm. Bacteria isolated from diseased fish were subjected to analysis using conventional bacteriological methods, a commercial bacterial identification test kit (API), an automated bacteria identification system known as Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), BD Phoenix™, and 16S rRNA sequence analysis. All isolated bacteria were identified as V. anguillarum by API 20E and conventional bacteriological method. These results have been confirmed with 16S rRNA sequence analysis. However, the isolated bacteria were identified as Grimontia hollisae (syn. Vibrio holisae) with the BD Phoenix system. Histologically, tissue damage such as melano-macrophage centers and necrosis in the kidney and spleen, hyperemia and mononuclear cell infiltration in the liver, as well as mononuclear cell infiltration on muscles, talengectiasis in the gill tissue was observed. In addition, it has been determined that the most effective antibiotic against the pathogen was enrofloxacin. When comparing all identification methods used for this pathogen causing tissue damage, it was demonstrated that the MALDI-TOF MS provides better results than other methods in terms of cost and identification time, and it could be used as an alternative to the conventional method to the identification of V. anguillarum.
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Enfermedades de los Peces , Oncorhynchus mykiss , ARN Ribosómico 16S , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Vibriosis , Vibrio , Animales , Vibrio/aislamiento & purificación , Vibrio/patogenicidad , Vibrio/efectos de los fármacos , Oncorhynchus mykiss/microbiología , Vibriosis/veterinaria , Vibriosis/microbiología , Enfermedades de los Peces/microbiología , ARN Ribosómico 16S/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Antibacterianos/farmacología , FilogeniaRESUMEN
Rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) is an important economic cold-water fish that is susceptible to heat stress. To date, the heat stress response in rainbow trout is more widely understood at the transcriptional level, while little research has been conducted at the translational level. To reveal the translational regulation of heat stress in rainbow trout, in this study, we performed a ribosome profiling assay of rainbow trout liver under normal and heat stress conditions. Comparative analysis of the RNA-seq data with the ribosome profiling data showed that the folding changes in gene expression at the transcriptional level are moderately correlated with those at the translational level. In total, 1213 genes were significantly altered at the translational level. However, only 32.8% of the genes were common between both levels, demonstrating that heat stress is coordinated across both transcriptional and translational levels. Moreover, 809 genes exhibited significant differences in translational efficiency (TE), with the TE of these genes being considerably affected by factors such as the GC content, coding sequence length, and upstream open reading frame (uORF) presence. In addition, 3468 potential uORFs in 2676 genes were identified, which can potentially affect the TE of the main open reading frames. In this study, Ribo-seq and RNA-seq were used for the first time to elucidate the coordinated regulation of transcription and translation in rainbow trout under heat stress. These findings are expected to contribute novel data and theoretical insights to the international literature on the thermal stress response in fish.
Asunto(s)
Respuesta al Choque Térmico , Hígado , Oncorhynchus mykiss , Biosíntesis de Proteínas , Ribosomas , Análisis de Secuencia de ARN , Animales , Oncorhynchus mykiss/genética , Respuesta al Choque Térmico/genética , Ribosomas/metabolismo , Ribosomas/genética , Biosíntesis de Proteínas/genética , Hígado/metabolismo , Regulación de la Expresión Génica , Transcripción Genética , Perfilación de la Expresión Génica , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Sistemas de Lectura Abierta/genética , Transcriptoma , Perfilado de RibosomasRESUMEN
Infections pose a challenge for the fast growing aquaculture sector. Glycosphingolipids are cell membrane components that pathogens utilize for attachment to the host to initiate infection. Here, we characterized rainbow trout glycosphingolipids from five mucosal tissues using mass spectrometry and nuclear magnetic resonance and investigated binding of radiolabeled Aeromonas salmonicida to the glycosphingolipids on thin-layer chromatograms. 12 neutral and 14 acidic glycosphingolipids were identified. The glycosphingolipids isolated from the stomach and intestine were mainly neutral, whereas glycosphingolipids isolated from the skin, gills and pyloric caeca were largely acidic. Many of the acidic structures were poly-sialylated with shorter glycan structures in the skin compared to the other tissues. The sialic acids found were Neu5Ac and Neu5Gc. Most of the glycosphingolipids had isoglobo and ganglio core chains, or a combination of these. The epitopes on the rainbow trout glycosphingolipid glycans differed between epithelial sites leading to differences in pathogen binding. A major terminal epitope was fucose, that occurred attached to GalNAc in a α1-3 linkage but also in the form of HexNAc-(Fuc-)HexNAc-R. A. salmonicida were shown to bind to neutral glycosphingolipids from the gill and intestine. This study is the first to do a comprehensive investigation of the rainbow trout glycosphingolipids and analyze binding of A. salmonicida to glycosphingolipids. The structural information paves the way for identification of ways of interfering in pathogen colonization processes to protect against infections in aquaculture and contributes towards understanding A. salmonicida infection mechanisms.
Asunto(s)
Aeromonas salmonicida , Glicoesfingolípidos , Oncorhynchus mykiss , Animales , Oncorhynchus mykiss/microbiología , Oncorhynchus mykiss/metabolismo , Aeromonas salmonicida/metabolismo , Aeromonas salmonicida/química , Glicoesfingolípidos/metabolismo , Glicoesfingolípidos/química , Membrana Mucosa/microbiología , Membrana Mucosa/metabolismoRESUMEN
The generic term "Gill disease" refers to a wide range of disorders that affect the gills and severely impact salmonid aquaculture systems worldwide. In rainbow trout freshwater aquaculture, various etiological agents causing gill diseases have been described, particularly Flavobacterium and Amoeba species, but research studies suggest a more complex and multifactorial aetiology. Here, a cohort of rainbow trout affected by gill disease is monitored both through standard laboratory techniques and 16S rRNA Next-Generation Sequencing (NGS) analysis during a natural disease outbreak and subsequent antibiotic treatment with Oxytetracycline. NGS results show a clear clustering of the samples between pre- and post-treatment based on the microbial community of the gills. Interestingly, the three main pathogenic bacteria species in rainbow trout (Yersinia ruckeri, Flavobacterium psychrophilum, and Flavobacterium branchiophilum) appear to be weak descriptors of the diversity between pre-treatment and post-treatment groups. In this study, the dynamics of the gill microbiome during the outbreak and subsequent treatment are far more complex than previously reported in the literature, and environmental factors seem of the utmost importance in determining gill disease. These findings present a potential novel perspective on the diagnosis and management of gill diseases, showing the limitations of conventional laboratory methodologies in elucidating the complexity of this disease in rainbow trout. To the authors' knowledge, this work is the first to describe the microbiome of rainbow trout gills during a natural outbreak and subsequent antibiotic treatment. The results of this study suggest that NGS can play a critical role in the analysis and comprehension of gill pathology. Using NGS in future research is highly recommended to gain deeper insights into such diseases correlating gill's microbiome with other possible cofactors and establish strong prevention guidelines.
Asunto(s)
Acuicultura , Brotes de Enfermedades , Enfermedades de los Peces , Flavobacterium , Branquias , Microbiota , Oncorhynchus mykiss , ARN Ribosómico 16S , Animales , Oncorhynchus mykiss/microbiología , Branquias/microbiología , Enfermedades de los Peces/microbiología , Enfermedades de los Peces/epidemiología , Flavobacterium/genética , Flavobacterium/aislamiento & purificación , Flavobacterium/patogenicidad , Brotes de Enfermedades/veterinaria , ARN Ribosómico 16S/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Yersinia ruckeri/genética , Infecciones por Flavobacteriaceae/veterinaria , Infecciones por Flavobacteriaceae/microbiología , Infecciones por Flavobacteriaceae/epidemiología , Oxitetraciclina/uso terapéutico , Oxitetraciclina/farmacología , Antibacterianos/farmacología , Antibacterianos/uso terapéuticoRESUMEN
The frequency of detection and concentrations of bifenthrin, a pyrethroid insecticide, in the waterways inhabited by the endangered species, steelhead trout (Oncorhynchus mykiss), has become a significant concern for regulatory agencies. Endocrine disruption has been observed with estrogenic and anti-estrogenic responses in fish species at different life stages. Since several studies have indicated alterations in dopaminergic signaling associated with endocrine responses, juvenile steelhead were exposed to environmentally relevant concentrations of 60 or 120 ng/L bifenthrin for two weeks. Fish brains were assessed for dopamine levels and the expression of genes involved in dopaminergic and estrogenic processes, such as catechol-o-methyltransferase (comt) and monoamine oxidase (mao). Vitellogenin (vtg) and estrogenic receptors (ERα1, ERß1, and ERß2) were also evaluated in livers of the animals. Dopamine concentrations were significantly higher in fish brains following bifenthrin exposure. Consistent with a reduction in dopamine clearance, there was a significant decrease in the mRNA expression of comt with increased bifenthrin concentration. Hepatic expression of ERα1 and ERß2 mRNA was significantly decreased with increased bifenthrin concentration. These data support the possible mechanism of bifenthrin altering the dopaminergic pathway at low ng/L concentrations, in juvenile steelhead, which could interfere with endocrine feedback loops. These findings support the need for and importance of identifying species and life stage differences in pesticide modes of action to reduce uncertainties in risk assessments.
Asunto(s)
Encéfalo , Dopamina , Insecticidas , Oncorhynchus mykiss , Piretrinas , Contaminantes Químicos del Agua , Animales , Piretrinas/toxicidad , Contaminantes Químicos del Agua/toxicidad , Oncorhynchus mykiss/metabolismo , Dopamina/metabolismo , Insecticidas/toxicidad , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Catecol O-Metiltransferasa/genética , Catecol O-Metiltransferasa/metabolismo , Disruptores Endocrinos/toxicidad , Receptores de Estrógenos/metabolismo , Receptores de Estrógenos/genética , Proteínas de Peces/metabolismo , Proteínas de Peces/genética , Vitelogeninas/metabolismo , Vitelogeninas/genéticaRESUMEN
Triploid Oncorhynchus mykiss is an important economic fish worldwide. Fishing stress can affect its growth and meat quality. This study first explored the effects of fishing stress on fatty acid and amino acid in triploid O. mykiss. Results showed fishing stress significantly reduced the content of docosadienoic acid, Gly, Arg, and DAA (P < 0.05). Targeted lipidomics analysis furthered suggested that some lipid molecules belonging to TG, DG, PC, Cer, ChE, and So were significantly up-regulated; while some lipid molecules belonging to Cer, LPE, LPC, PS, PC, and SM were significantly down-regulated, suggesting an accelerated glycolipid metabolism. Eventually, the glycolipid metabolism-related enzyme activity and gene expressions were examined, and the results indicated that O. mykiss was anti-oxidative stress by affecting relevant glycolipid metabolism signaling pathways and participating in cellular redox homeostasis. Findings of this study provide a theoretical foundation for further investigation into the mechanisms through which fishing stress affects O. mykiss.
Asunto(s)
Aminoácidos , Ácidos Grasos , Glucolípidos , Oncorhynchus mykiss , Animales , Oncorhynchus mykiss/metabolismo , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/crecimiento & desarrollo , Aminoácidos/metabolismo , Aminoácidos/análisis , Glucolípidos/metabolismo , Ácidos Grasos/metabolismo , Ácidos Grasos/química , Triploidía , Estrés Fisiológico , Metabolismo de los Lípidos , Explotaciones PesquerasRESUMEN
The aim of our study was to investigate the effects of cyanobacterial metabolites: microcystin-LR (MC-LR) anabaenopeptin-A (ANA-A), cylindrospermopsin (CYL), their binary and ternary mixtures on rainbow trout (Oncorhynchus mykiss) gill (RTgill-W1) cell line. We determined the following cell parameters: Hoechst and propidium iodide (PI) double staining, intracellular ATP level with luminometric assay, glutathione level with ThiolTracker Violet®- glutathione detection reagent and cytoskeletal F-actin fluorescence. The results showed that although reduction of Hoechst fluorescence was observed in both binary and ternary combinations of cyanobacterial metabolites, the mixture of MC-LR + ANA-A + CYL was the most potent inhibitor (EC50 = 148 nM). PI fluorescence and ATP levels were more increased in the cells exposed to the mixtures than those exposed to the individual metabolites with synergistic toxic changes suggesting apoptosis as the mechanism of cell death. Reduced glutathione level was also decreased in cells exposed both to single metabolites and their mixtures with the highest decrease and synergistic effects at 334 nM MC-LR+334 nM ANA-A+ 334 nM CYL suggesting induction oxidative stress by the tested compounds. Reduction of F-actin fluorescence was found in the cells from all of the groups exposed to individual metabolites and their mixtures, however the highest level of inhibition showed the binary MC-LR + CYL and the ternary MC-LR + ANA-A + CYL with synergistic interactions. The study suggests that in natural conditions fish gill cells may be very sensitive to individual cyanobacterial metabolites and more prone to their binary and ternary mixtures.
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Alcaloides , Toxinas de Cianobacterias , Cianobacterias , Toxinas Marinas , Microcistinas , Oncorhynchus mykiss , Uracilo , Microcistinas/metabolismo , Animales , Alcaloides/farmacología , Uracilo/análogos & derivados , Línea Celular , Cianobacterias/metabolismo , Oncorhynchus mykiss/metabolismo , Branquias/metabolismo , Branquias/efectos de los fármacos , Glutatión/metabolismo , Toxinas Bacterianas , Adenosina Trifosfato/metabolismo , Apoptosis/efectos de los fármacos , Péptidos Cíclicos/farmacologíaRESUMEN
Fresh water sources, including lakes, such as the Great Lakes, are some of the most important ecosystems in the world. Despite the importance of these lakes, there is increasing concern about the presence of per- and polyfluoroalkyl substances (PFAS)âamong the most prevalent contaminants of our timeâdue to the ability of PFAS to bioaccumulate and persist in the environment, as well as to its linkages to detrimental human and animal health effects. In this study, PFAS exposure on rainbow trout (Oncorhynchus mykiss) is examined at the molecular level, focusing on the impact of PFAS binding on the alpha (α) and beta (ß) estrogen receptors (ERs) using molecular dynamics simulations, binding free energy calculations, and structural analysis. ERs are involved in fundamental physiological processes, including reproductive system development, muscle regeneration, and immunity. This study shows that PFAS binds to both the estrogen α and estrogen ß receptors, albeit via different binding modes, due to a modification of an amino acid in the binding site as a result of a reorientation of residues in the binding pocket. As ER overactivation can occur through environmental toxins and pollutants, this study provides insights into the influence of different types of PFAS on protein function.
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Oncorhynchus mykiss , Receptores de Estrógenos , Contaminantes Químicos del Agua , Animales , Oncorhynchus mykiss/metabolismo , Receptores de Estrógenos/metabolismo , Contaminantes Químicos del Agua/metabolismo , Simulación de Dinámica Molecular , Receptor alfa de Estrógeno/metabolismoRESUMEN
There is a consensus that electroneutral Na+/H+ exchangers (NHEs) are important in branchial Na+ uptake in freshwater fish. There is also widespread belief, based on mammalian data, that EIPA [5-(N-ethyl-N-isopropyl)-amiloride]], and HMA [5-(N,N-hexamethylene)-amiloride)] are more potent and specific in blocking Na+ uptake than amiloride. We evaluated this idea by testing the three drugs at 10-7 to 10-4 M, i.e. 0.1 to 100 µM in two model species, rainbow trout (Oncorhynchus mykiss) and goldfish (Carassius auratus), using 22Na+ to measure unidirectional Na+ influx and efflux rates. In both species, the potency order for inhibiting unidirectional Na+ influx was HMA > amiloride > EIPA (IC50 values in the 10-70 µM range), very different from in mammals. At 100 µM, all three drugs inhibited Na+ influx by >90% in both species, except for amiloride in goldfish (65%). However, at 60-100 µM, all three drugs also stimulated unidirectional Na+ efflux rates, indicating non-specific effects. In trout, HMA and EIPA caused significant increases (2.1- to 2.3-fold) in efflux rates, whereas in goldfish, significant efflux elevations were greater (3.1- to 7.2-fold) with all three drugs. We conclude that the inhibitory potency profile established in mammals does not apply to the NHEs in fish gills, that non-specific effects on Na+ efflux rates are a serious concern, and that EIPA and HMA offer no clear benefits in terms of potency or specificity. Considering its much lower cost, we recommend amiloride as the drug of choice for in vivo experiments on freshwater fishes.
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Amilorida , Carpa Dorada , Sodio , Animales , Amilorida/farmacología , Amilorida/análogos & derivados , Carpa Dorada/metabolismo , Sodio/metabolismo , Branquias/metabolismo , Branquias/efectos de los fármacos , Oncorhynchus mykiss/metabolismo , Agua Dulce , Intercambiadores de Sodio-Hidrógeno/metabolismo , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Transporte Iónico/efectos de los fármacos , Trucha/metabolismoRESUMEN
Aeromonas salmonicida is an opportunistic pathogen with relevance for aquaculture. Fish epithelia are covered by a mucus layer, composed mainly by highly glycosylated mucins, which are the first point of contact between fish and pathogens. Quorum sensing (QS), a bacterial communication mechanism through secreted autoinducer signals that governs gene expression, influences bacterial growth and virulence. The main A. salmonicida autoinducers are mediated by the luxS and asaI genes, corresponding to inter- and intraspecies communication, respectively. The aim of this study was to determine the effect of the mucins that pathogens encounter during colonization of the gill and skin on A. salmonicida QS. We found that expression of A. salmonicida asaI, but not luxS, was increased after culture at 20 °C compared to 10 °C. Rainbow trout gill and skin mucins up-regulated asaI expression 2-fold but down-regulated luxS 10-fold. The downregulation of luxS was reflected by a reduction in autoinducer-2 secretion. Mucins isolated from skin had a stronger inhibitory effect than mucins isolated from gills on both luxS expression and A1-2 secretion, consistent with a higher relative abundance of N-Acetylneuraminic acid on skin mucins than on gill mucins. Reduction of AI-2 production by mucins or luxS-deletion lead to a reduced A. salmonicida auto-aggregation. Furthermore, after colonization of the gill, luxS was down regulated whereas asaI expression was upregulated. Both in vivo and in vitro, the expression of luxS and asaI were thus differentially regulated, frequently in an inverse manner. The strong AI-2 inhibiting effect of the skin mucins is likely part of the mucin-based defense against pathogens.
Asunto(s)
Aeromonas salmonicida , Homoserina , Mucinas , Oncorhynchus mykiss , Percepción de Quorum , Animales , Oncorhynchus mykiss/inmunología , Aeromonas salmonicida/fisiología , Mucinas/genética , Mucinas/metabolismo , Homoserina/análogos & derivados , Liasas de Carbono-Azufre/genética , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Proteínas Bacterianas/genética , Lactonas , Piel/inmunología , Piel/microbiología , Branquias/inmunología , Branquias/metabolismoRESUMEN
The growth hormone (GH)-insulin-like growth factor-1 (IGF-1) system regulates skeletal muscle growth and function. GH has a major function of targeting the liver to regulate IGF-1 production and release, and IGF-1 mediates the primary anabolic action of GH on growth. However, skeletal muscle is a target tissue of GH as evidenced by dynamic GH receptor expression, but it is unclear if GH elicits any direct actions on extrahepatic tissues as it is difficult to distinguish the effects of IGF-1 from GH. Fish growth regulation is complex compared to mammals, as genome duplication events have resulted in multiple isoforms of GHs, GHRs, IGFs, and IGFRs expressed in most fish tissues. This study investigated the potential for GH direct actions on fish skeletal muscle using an in vitro system, where rainbow trout myogenic precursor cells (MPCs) were cultured in normal and serum-deprived media, to mimic in vivo fasting conditions. Fasting reduces IGF-1 signaling in the muscle, which is critical for disentangling the roles of GH from IGF-1. The direct effects of GH were analyzed by measuring changes in myogenic proliferation and differentiation genes, as well as genes regulating muscle growth and proteolysis. This study provides the first in-depth analysis of the direct actions of GH on serum-deprived fish muscle cells in vitro. Data suggest that GH induces the expression of markers for proliferation and muscle growth in the presence of serum, but all observed GH action was blocked in serum-deprived conditions. Additionally, serum deprivation alone reduced the expression of several proliferation and differentiation markers, while increasing growth and proteolysis markers. Results also demonstrate dynamic gene expression response in the presence of GH and a JAK inhibitor in serum-provided but not serum-deprived conditions. These data provide a better understanding of GH signaling in relation to serum in trout muscle cells in vitro.
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Hormona del Crecimiento , Factor I del Crecimiento Similar a la Insulina , Músculo Esquelético , Oncorhynchus mykiss , Animales , Oncorhynchus mykiss/metabolismo , Oncorhynchus mykiss/genética , Hormona del Crecimiento/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/efectos de los fármacos , Transducción de Señal , Células Cultivadas , Proteínas de Peces/metabolismo , Proteínas de Peces/genética , Receptores de Somatotropina/metabolismo , Receptores de Somatotropina/genéticaRESUMEN
Current prophylactic and disease control measures in aquaculture highlight the need of alternative strategies to prevent disease and reduce antibiotic use. Mucus covered mucosal surfaces are the first barriers pathogens encounter. Mucus, which is mainly composed of highly glycosylated mucins, has the potential to contribute to disease prevention if we can strengthen this barrier. Therefore, aim of this study was to develop and characterize fish in vitro mucosal surface models based on commercially available cell lines that are functionally relevant for studies on mucin regulation and host-pathogen interactions. The rainbow trout (Oncorhynchus mykiss) gill epithelial cell line RTgill-W1 and the embryonic cell line from Chinook salmon (Oncorhynchus tshawytscha) CHSE-214 were grown on polycarbonate membrane inserts and chemically treated to differentiate the cells into mucus producing cells. RTGill-W1 and CHSE-214 formed an adherent layer at two weeks post-confluence, which further responded to treatment with the γ-secretase inhibitor DAPT and prolonged culture by increasing the mucin production. Mucins were metabolically labelled with N-azidoacetylgalactosamine 6 h post addition to the in vitro membranes. The level of incorporated label was relatively similar between membranes based on RTgill-W1, while larger interindividual variation was observed among the CHSE in vitro membranes. Furthermore, O-glycomics of RTgill-W1 cell lysates identified three sialylated O-glycans, namely Galß1-3(NeuAcα2-6)GalNAcol, NeuAcα-Galß1-3GalNAcol and NeuAcα-Galß1-3(NeuAcα2-6)GalNAcol, resembling the glycosylation present in rainbow trout gill mucin. These glycans were also present in CHSE-214. Additionally, we demonstrated binding of the fish pathogen A. salmonicida to RTgill-W1 and CHSE-214 cell lysates. Thus, these models have similarities to in vivo mucosal surfaces and can be used to investigate the effect of pathogens and modulatory components on mucin production.
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Interacciones Huésped-Patógeno , Mucinas , Oncorhynchus mykiss , Animales , Mucinas/metabolismo , Oncorhynchus mykiss/metabolismo , Línea Celular , Membrana Mucosa/metabolismo , Salmón/metabolismo , Branquias/metabolismo , Células Epiteliales/metabolismo , Moco/metabolismoRESUMEN
Infectious hematopoietic necrosis virus (IHNV) severely and lethally infects salmonid fish, including Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) worldwide. Rapid and accurate viral detection is crucial for preventing pathogen spread and minimizing damage. Although several IHNV detection assays have been developed, their analytical and diagnostic performances have not been evaluated and field usability assessments have not been completely validated. Here, we developed a reverse-transcription cross-priming amplification-based lateral flow assay (RT-CPA-LFA) and validated its diagnostic performance. To detect the IHNV, primers were designed based on the consensus sequence of the nucleocapsid (N) gene. Notably, when combined with a lateral flow dipstick, it could visualize the IHNV amplification products within 5â¯min and the detection limit of the developed RT-CPA-LFA was 3.28×105 copies/µL. The diagnostic sensitivity and specificity in fish samples (n=140) were 98.88â¯% and 96.08â¯%, respectively. Moreover, the IHNV detection rate by RT-CPA-LFA in dead rainbow trout artificially injected with the virus was 100â¯%, consistent with to the results obtained from second conventional and real-time PCR, indicating its applicability for rapid IHNV detection and presumptive IHN diagnosis during the endemic period.
Asunto(s)
Cartilla de ADN , Enfermedades de los Peces , Virus de la Necrosis Hematopoyética Infecciosa , Oncorhynchus mykiss , Infecciones por Rhabdoviridae , Sensibilidad y Especificidad , Virus de la Necrosis Hematopoyética Infecciosa/genética , Virus de la Necrosis Hematopoyética Infecciosa/aislamiento & purificación , Animales , Infecciones por Rhabdoviridae/veterinaria , Infecciones por Rhabdoviridae/diagnóstico , Infecciones por Rhabdoviridae/virología , Enfermedades de los Peces/diagnóstico , Enfermedades de los Peces/virología , Oncorhynchus mykiss/virología , Cartilla de ADN/genética , Salmo salar/virología , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas de Amplificación de Ácido Nucleico/veterinaria , Transcripción Reversa , Técnicas de Diagnóstico Molecular/métodosRESUMEN
Residues of human pharmaceuticals are widely detected in surface waters and can be taken up by and bioaccumulate in aquatic organisms, especially fish. One of the key challenges in assessing the bioaccumulation potential of ionizable organic compounds, such as the pharmaceuticals, is the lack of empirical data for biotransformation. In the present study, we assessed the in vitro intrinsic clearances (CLINT) of twelve pharmaceuticals, individually and some additionally as mixtures, in rainbow trout (Oncorhynchus mykiss) liver S9 fractions (RT-S9) adhering to the OECD test guidance 319B. The test substances included four anti-inflammatory agents (diclofenac, ibuprofen, ketoprofen, naproxen), seven antidepressants/antipsychotics (citalopram, haloperidol, levomepromazine, mirtazapine, risperidone, sertraline, venlafaxine) and the O-desmethyl metabolite of venlafaxine. Quantifiable intrinsic clearances were detected for diclofenac, ibuprofen, naproxen, levomepromazine, and sertraline. Apart from diclofenac, the in vitro clearances of the other four pharmaceuticals were shown to be critically dependent on the cytochrome P450 (CYP) metabolism. Therefore, we also determined the half-maximal inhibitory concentrations (IC50) of the same twelve pharmaceuticals toward CYP1A-like (7-ethoxyresorufin-O-deethylation, EROD) and CYP3A-like (benzyloxy-4-trifluoromethylcoumarin-O-debenzyloxylation, BFCOD) activities in RT-S9 using IC50 shift assay. As a result, levomepromazine and sertraline were identified as the most potent inhibitors of both EROD and BFCOD activity (unbound IC50 < 10 µM each), followed by citalopram and haloperidol (10 µM < IC50 < 100 µM). Additionally, mirtazapine was a selective EROD inhibitor (IC50 â¼ 30 µM). The inhibitory impacts of haloperidol and sertraline were indicatively time dependent. Finally, we carried out intrinsic clearance assays with mixtures of diclofenac, ibuprofen, naproxen, levomepromazine, and sertraline to examine the impacts of EROD and BFCOD inhibitions on their in vitro CLINT in RT-S9. Our in vitro data suggests that the intrinsic clearances of ibuprofen, levomepromazine, and sertraline in rainbow trout can be significantly reduced as the result of P450 inhibition by pharmaceutical mixtures, whereas the clearances of diclofenac and naproxen are less impacted.
Asunto(s)
Antiinflamatorios , Antidepresivos , Antipsicóticos , Hígado , Oncorhynchus mykiss , Contaminantes Químicos del Agua , Animales , Oncorhynchus mykiss/metabolismo , Contaminantes Químicos del Agua/toxicidad , Hígado/efectos de los fármacos , Hígado/metabolismo , Antiinflamatorios/farmacología , Microsomas Hepáticos/metabolismo , Microsomas Hepáticos/efectos de los fármacosRESUMEN
This study aimed to investigate the protective effects of Allium jesdianum essential oil (AJEO) in decreasing cypermethrin toxicity for rainbow trout. First, the safety of the 0%, 0.5%, 1%, and 1.5% AJEO supplements was assayed after 60 days. Then, the protective effects of AJEO were studied on fish exposed to 12.5% 96h LC50 cypermethrin after 14 days. Results showed that 1 and 1.5% AJEO administration enhanced protease and lipase activities in the intestine and improved growth performance. Moreover, feeding fish with 1 and 1.5% AJEO increased catalase (CAT) and superoxide dismutase activities (SOD) and decreased malondialdehyde (MDA). Also, AJEO increased glutathione peroxidase (GPx) activity in serum. However, exposure to cypermethrin significantly decreased these enzyme activities and increased MDA. The oxidative biomarkers remained normal in fish fed with AJEO after exposure to cypermethrin. The administration of 1 and 1.5% AJEO significantly decreased cortisol and glucose levels, alkaline phosphatase (ALP), lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase activities. Although exposure to cypermethrin significantly increased these biochemical biomarkers, AJEO could adjust them. A significant effect of 1% AJEO on total protein and globulin was observed before and after exposure to cypermethrin. Exposure to cypermethrin decreased all immunological parameters in the serum and mucus. However, administration of 1% AJEO increased protease, lysozyme (LYS) activities, total immunoglobulin (Ig), complement C3 and C4, and nitroblue tetrazolium (NBT) in the serum and ALP, LYS, protease activities and Ig in mucus. In conclusion, results showed that AJEO could potentially decrease the toxicity effects of cypermethrin in fish.
Asunto(s)
Aceites Volátiles , Oncorhynchus mykiss , Piretrinas , Contaminantes Químicos del Agua , Animales , Piretrinas/toxicidad , Contaminantes Químicos del Agua/toxicidad , Aceites Volátiles/toxicidad , Insecticidas/toxicidad , Estrés Oxidativo/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Catalasa/metabolismo , Malondialdehído/metabolismo , Glutatión Peroxidasa/metabolismoRESUMEN
Yersiniosis, caused by Yersinia ruckeri, has become the most common disease in farmed rainbow trout Oncorhynchus mykiss in Peru, affecting Puno and Junín Regions, important aquaculture areas in the country. Florfenicol (FLO) and oxytetracycline (OXY) are the antimicrobials most frequently used to mitigate losses attributed to this pathogen. This study presents an analysis of the susceptibility patterns of 60 Y. ruckeri isolates (30 isolates each from Puno and Junín), including the type strain CECT 4319T and the strains CECT 955 and CECT 956, against FLO and OXY. Minimum inhibitory concentrations (MICs) were determined following the guideline for standard broth dilution method published by the Clinical and Laboratory Standards Institute. MIC results ranged from 4.0 to 8.0 µg ml-1 for FLO and 0.5 to 4.0 µg ml-1 for OXY. Normalized resistance interpretation (NRI) analysis identified epidemiological cut-off values of ≤16.0 µg ml-1 for FLO and ≤4.0 µg ml-1 for OXY. All Peruvian isolates, including the collection strains, were categorized as wild-type for both antimicrobials. Even though the number of Y. ruckeri isolates with MIC values of 8 µg ml-1 for FLO is more than double in Puno than in Junín (15 vs. 7 isolates), the NRI analysis showed the same epidemiological cutoff of 16 µg ml-1; while for OXY, it was 4.0 µg ml-1 for Puno and 2.0 µg ml-1 for Junín. This study establishes the basis for monitoring susceptibility to FLO and OXY in new Y. ruckeri isolates in Peruvian rainbow trout farming.
Asunto(s)
Antibacterianos , Enfermedades de los Peces , Pruebas de Sensibilidad Microbiana , Oxitetraciclina , Tianfenicol , Yersinia ruckeri , Antibacterianos/farmacología , Tianfenicol/análogos & derivados , Tianfenicol/farmacología , Yersinia ruckeri/efectos de los fármacos , Perú/epidemiología , Oxitetraciclina/farmacología , Animales , Enfermedades de los Peces/microbiología , Farmacorresistencia Bacteriana , Yersiniosis/veterinaria , Yersiniosis/microbiología , Oncorhynchus mykissRESUMEN
Ichthyophonosis is a disease caused by the mesomycetozoean parasite Ichthyophonus hoferi that affects a variety of fish species, including rainbow trout Oncorhynchus mykiss Walbaum. This disease is characterized by granulomatous lesions and necrosis in various organs, which can have severe impacts on the health and welfare of the fish. Ichthyophonosis has been found in several parts of the world, including Europe, and is a significant concern in the aquaculture industry and for populations of wild marine fishes. The rainbow trout is a widely cultured salmonid species in many countries, including Serbia. Although the presence of I. hoferi in rainbow trout has been reported in several countries, it has never been documented in Serbia. In this article, we report the first case of ichthyophonosis in rainbow trout in Serbia.
Asunto(s)
Acuicultura , Enfermedades de los Peces , Infecciones por Mesomycetozoea , Mesomycetozoea , Oncorhynchus mykiss , Animales , Enfermedades de los Peces/parasitología , Enfermedades de los Peces/epidemiología , Serbia/epidemiología , Infecciones por Mesomycetozoea/epidemiología , Infecciones por Mesomycetozoea/parasitología , Mesomycetozoea/aislamiento & purificaciónRESUMEN
An in vitro study using rainbow trout spermatozoa was designed to evaluate the toxic effects of different concentrations of captan (CPT), mancozeb (MCZ), and azoxystrobin (AZX) fungicides on motility parameters, lipid peroxidation, SOD activity, total antioxidant capacity (TAC), and DPPH inhibition. Moreover, changes in fatty acids profiles caused by the fungicides were determined for the first time. The results revealed that motility parameters, SOD activities, TAC values, and DPPH inhibitions decreased significantly while lipid peroxidation increased after ≥2 µg/L of CPT, ≥1 µg/L of MCZ, and ≥5 µg/L of AZX incubations for 2 h at 4 °C. Additionally, 10 µg/L CPT, 5 µg/L MCZ, and 200 µg/L AZX reduced motility to the 50 % level. Our results clearly demonstrated significant changes in the fatty acids profiles of spermatozoa exposed to these concentrations of the fungicides. The highest lipid peroxidation and the lowest monounsaturated and polyunsaturated saturated fatty acids (MUFA and PUFA, respectively) were detected in AZX. Even though the susceptibility of spermatozoa to oxidative damage is generally attributed to PUFA contents, the results of this study have represented that MUFA content could play a part in this tendency. Moreover, the lower concentration of MCZ reduced motility to the % 50 level while it deteriorated the fatty acids profile less than did AZX. Overall, the present study demonstrated that the detrimental effects of the fungicides on mitochondrial respiration and related enzymes have more priority than oxidative stress in terms of their toxicities on spermatozoa. It has also been suggested that fish spermatozoa are a good model for determining changes in the fatty acid profiles by fungicides, probably, by other pesticides and environmental contaminants as well.