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Los salmónidos, durante su desarrollo, pueden experimentar ocasionalmente deformaciones esqueléticas. Para su diagnóstico se emplean diversas metodologías, entre las que se incluyen radiografías, técnicas histológicas, diafanización con tinciones de alizarina y azul de alcián, así como el uso del microscopio electrónico de barrido (SEM), cada una con sus inherentes ventajas y desventajas. Este estudio tuvo como finalidad evaluar y comparar la eficacia de la tomografía microcomputarizada (Micro-CT) para el análisis anatómico, reconstruyendo tridimensionalmente las imágenes y contrastándolas con los resultados obtenidos mediante la técnica de diafanización. Se analizaron las aletas caudales de cinco ejemplares de salmón Oncorhynchus kisutch: dos sujetos a diafanización y tres procesados para análisis mediante Micro-CT utilizando el equipo BRUKER SkyScan 1272. La técnica de Micro-CT demostró superioridad en la resolución de las estructuras óseas, facilitando una exploración detallada de las variaciones morfológicas y la distribución de la densidad mineral. Este enfoque permitió identificar anomalías en la morfología y crecimiento de las últimas vértebras y lepidotriquias dorsales, así como una densidad incrementada en lepidotriquias dorsales malformadas. La mayor resolución proporcionada por la Micro-CT no solo potencia nuestra comprensión de la ontogenia piscícola y su adaptación a ambientes diversos, sino que además inaugura perspectivas innovadoras para el estudio de la evolución de las estrategias locomotoras y las respuestas adaptativas frente a cambios ambientales a través del tiempo.

SUMMARY: During their development, some species of salmonids may occasionally experience skeletal deformations. Several methodologies are currently being used for the diagnosis of such malformations, among which X-rays, histological techniques, diaphanization coupled either with Alizarin Red or Alcian Blue stains, as well as Scanning Electron Microscopy (SEM) can be mentioned. Each one of those methods presents inherent advantages and disadvantages. The purpose of this study was twofold: Firstly, to evaluate and compare the effectiveness of microcomputed tomography (Micro-CT) technology for anatomical analysis, three-dimensionally reconstructing the obtained images; and secondly, to contrast those images with the results obtained through the diaphanization technique. The caudal fins of five specimens of the Oncorhynchus kisutch salmon were analyzed: Two specimens were subjected to diaphanization and three were processed for Micro-CT analysis, using the BRUKER SkyScan 1272 equipment. The Micro-CT technology demonstrated superiority in the resolution of bone structures, facilitating a detailed exploration of morphological variations, as well as the distribution of mineral density. This experimental approach allowed us to identify anomalies in the morphology and growth of the last vertebrae and dorsal lepidotrichiae, as well as an increased mineral density in the malformed dorsal lepidotrichiae. The higher resolution provided by Micro-CT not only enhances our understanding of the fish ontogeny and its adaptation to diverse environments, but also opens innovative perspectives for the study of the evolution of locomotor strategies and adaptive responses to environmental changes.

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Tenacibaculum piscium, a gram-negative bacterium isolated from the skin ulcers of sea-farmed fish, has only been described in Norway. In the present study, we examined 16 Chilean Tenacibaculum isolates recovered from different organs in moribund and dead Atlantic salmon (Salmo salar), Rainbow trout (Oncorhynchus mykiss) and Coho salmon (Oncorhynchus kisutch) cultured at different fish farms between 2014 and 2018. The present study applied biochemical, phenotypic, fatty acid and whole-genome sequence-based analyses to confirm the taxonomic status of the Chilean isolates. The obtained results are the first to confirm the presence of T. piscium in Chile and in Coho salmon, thus extending the recognized geographical and species distribution of this bacterium. Subsequent bath-challenge assays in Atlantic salmon utilizing three T. piscium isolates obtained from different hosts resulted in low cumulative mortality (i.e. 0-35%), even after exposure to an unnaturally high concentration of bacterial cells (i.e. > 107 cells/ml). However, scale loss and frayed fins were observed in dead fish. In silico whole-genome analysis detected various genes associated with iron acquisition, encoding of the type IX secretion system and cargo proteins, resistance to tetracycline and fluoroquinolones and stress responses. These data represent an important milestone towards a better understanding on the genomic repertoire of T. piscium.

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Piscirickettsiosis is the most important bacterial disease in the Chilean salmon industry, which has borne major economic losses due to failure to control it. Cells use extracellular vesicles (EVs) as an inter-cellular communicators to deliver several factors (e.g., microRNAs) that may regulate the responses of other cells. However, there is limited knowledge about the identification and characterization of EV-miRNAs in salmonids or the effect of infections on these. In this study, Illumina sequencing technology was used to identify Coho salmon plasma EV-miRNAs upon Piscirickettsia salmonis infection at four different time points. A total of 118 novels and 188 known EV-miRNAs, including key immune teleost miRNAs families (e.g., miR-146, miR-122), were identified. A total of 245 EV-miRNAs were detected as differentially expressed (FDR < 5%) in terms of control, with a clear down-regulation pattern throughout the disease. KEGG enrichment results of EV-miRNAs target genes showed that they were grouped mainly in cellular, stress, inflammation and immune responses. Therefore, it is hypothesized that P. salmonis could potentially benefit from unbalanced modulation response of Coho salmon EV-miRNAs in order to promote a hyper-inflammatory and compromised immune response through the suppression of different key immune host miRNAs during the course of the infection, as indicated by the results of this study.

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Several members of the Mycobacterium genus cause invasive infections in humans and animals. According to a recent phylogenetic analysis, some strains of Mycobacterium salmoniphilum (Msal), which are the main culprit in bacterial outbreaks in freshwater fish aquaculture, have been assigned to a separate branch containing Mycobacterium franklinii (Mfra), another species that causes infections in humans. However, this genus is little studied in an aquaculture context. Here, we isolated four Mycobacterium spp. strains from freshwater cultures of Atlantic and coho salmon in Chile and performed whole-genome sequencing for deep genomic characterization. In addition, we described the gross pathology and histopathology of the outbreaks. Several bioinformatic analyses were performed using the genomes of these four Mycobacterium isolates in conjunction with those of Msal strains, four Msal-like strains, and one Mfra strains, plus 17 other publicly available Mycobacterium genomes. We found that three isolates are clustered into the Msal branch, whereas one isolate clustered with the Mfra/Msal-like strains. We further evaluated the presence of virulence and antimicrobial resistance genes and observed that the four isolates were closely related to the Msal and Msal-like taxa and carried several antimicrobial resistance and virulence genes that are similar to those of other pathogenic members of the Mycobacterium clade. Altogether, our characterization Msal and Msal-like presented here shed new light on the basis of mycobacteriosis provides quantitative evidence that Mycobacterium strains are a potential risk for aquaculture asetiological agents of emerging diseases, and highlight their biological scopes in the aquaculture industry.

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Surveillance of antibiotic resistance is of paramount importance for animal welfare and production. Despite aquaculture being a main source of animal protein, studies on antibiotic susceptibility in fish pathogens are scarce. Renibacterium salmoninarum, the aetiological agent of bacterial kidney disease (BKD), is one of the most common bacterial pathogens affecting salmon farming. In this work, we present an analysis of susceptibility patterns using determinations of minimum inhibitory concentration (MIC) for 65 field isolates, which were collected over seven years (2013-2019) from Atlantic salmon (Salmo salar) and coho salmon (Oncorhynchus kisutch) farms across southern Chile. The MIC protocol described by the Clinical Laboratory Standards Institute (CLSI) was used, but with microdilution instead of macrodilution and eight instead of four days of incubation. Two laboratories independently conducted analyses to provide data on the epidemiological cut-off values for R. salmoninarum to florfenicol, oxytetracycline and erythromycin. By using two calculation methods, our results provide evidence for an evolving subpopulation of non-wild-type isolates for the macrolide erythromycin, which is consistent with the respective treatment frequencies prescribed against BKD. Contrasting with what was expected, R. salmoninarum isolates were most susceptible to florfenicol and oxytetracycline, both of which are widely used antibiotics currently used in the Chilean salmon industry. The presented findings can serve as a reference for national or international antibiotic surveillance programmes, for both MIC interpretation and to identify emerging resistance to the conventional drugs used in BKD management. Finally, our results indicate that an 8-day incubation period for establishing MIC values of R. salmoninarum should be considered in a future revision of the CLSI guidelines.

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This study aimed at estimating parameters representing between-farm transmission of Salmonid Rickettsial Septicaemia (SRS) in Chile, and developing and validating simulation models to predict weekly spread of SRS between farms in Los Lagos (Region 10), using InterSpread Plus. The model parameters were estimated by analyses of the historical SRS outbreak data. The models incorporated time and distance-dependent transmission kernels, representing the probabilities of waterborne spread of SRS between farms. Seven candidate transmission kernels were estimated, with varying maximum distance of between-farm SRS spread (15-60 km). Farms were categorized by size (small; medium; large) and species (Coho salmon; Atlantic salmon; rainbow trout). The time that it took a farm to recover from infection was parameterized to be shortest for small Coho farms (median: 7 weeks), followed by medium and large Coho farms (median: 25 weeks), Atlantic salmon farms (median: 42 weeks, any size) and rainbow trout farms (median: 43 weeks, any size). The relative infectiousness parameters of rainbow trout farms were 1.5-6.3 times that of Coho or Atlantic salmon, or those of large farms was 1.3-4.2 times that of small or medium farms. The models predicted SRS prevalence in Region 10 between 2013 and 2015 (79 weeks) with 76.5%-93.0% overall accuracy. The model with a transmission kernel of <20 km (P20) achieved a maximum overall accuracy (93.0%). Within each neighbourhood, the accuracy of P20 varied between 32.4% and 88.1%; 13/20 neighbourhoods had a reasonable temporal agreement between the simulated and actual dynamics of SRS (within 5th-95th percentiles), but 5/20 neighbourhoods underestimated and 2/20 overestimated the SRS spread. The model could be used for evaluation of semi-global control policies in Region 10, while addition of other factors such as seasonality, ocean currents, and movement of infected fish may improve the model performance at a finer scale.

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The success and sustainability of Chilean aquaculture largely depends on the control of endemic and emerging pathogens, including several species of the genus Tenacibaculum. Tenacibaculum dicentrarchi and "Tenacibaculum finnmarkense" have been detected and confirmed in Chilean Atlantic salmon (Salmo salar). However, no outbreaks of tenacibaculosis in rainbow trout (Oncorhynchus mykiss) or coho salmon (Oncorhynchus kisutch) have been reported, either in Chile or globally. The aims of this study were to determine whether the mortalities recorded for rainbow trout and coho salmon from five marine fish farms located in the Los Lagos, Aysén, and Magallanes Regions could be caused by Tenacibaculum spp. The diseased fish exhibited cutaneous haemorrhages, tail and peduncle rots, and damage on the mouth and tongue. Microbiological analysis of infected external tissues yielded 13 bacterial isolates. The isolates were identified as members of the genus Tenacibaculum through biochemical analysis (e.g. Gram-stain negative, straight rods, filamentous cells and motile by gliding), but differences existed in biochemical results, making species-level identification through biomolecular tools essential. The 16S rRNA analysis found that the majority of isolates were more closely related to "T. finnmarkense" than T. dicentrarchi, while the phylogenetic trees resulting from multilocus sequence data recovered the four main clades (clades I to IV) identified by Olsen et al. (2017, Veterinary Microbiology, 205, 39). This is the first documented occurrence of clinical tenacibaculosis in farmed rainbow trout and coho salmon globally, and it extends the known host distribution of this pathogen in Chile. Moreover, we confirm the presence of Tenacibaculum species in the Chilean Patagonia. These findings highlight the importance of establishing preventative measures to minimize the spread of this disease within the Chilean marine aquaculture industry, as well as the need for monitoring initiatives worldwide in these farmed fish species.

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Chile is currently the second largest producer of farmed salmon worldwide, but Flavobacterium psychrophilum, as one of the most detrimental pathogens, is responsible for major losses during the freshwater culturing step in salmonid fish farms. An antigenic study conducted 10 years ago reported four serological groups using 20 F. psychrophilum Chilean strains. To reduce disease outbreaks and to develop vaccine candidates, antigenic knowledge needs to be regularly updated using a significant number of additional recent F. psychrophilum isolates. The present study aimed at investigating the serological diversity of 118 F. psychrophilum isolates collected between 2006 and 2018 from farmed Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch). The current study supports an expansion of the known antigenic groups in Chile from 4 to 14. However, the use of the slide-agglutination technique for serotyping is costly, is labour-intensive and requires significant technical expertise. Addressing these points, the mPCR-based procedure was a very useful tool for serotyping the collected Chilean F. psychrophilum isolates. This technique revealed the presence of diverse mPCR serotypes (i.e. types 0, 1, 2 and 4). Therefore, mPCR should be employed to select the bacterial strain(s) for vaccine development and to conduct follow-up, selective breeding or epidemiological surveillance in Chilean fish farms. Given the presented findings, changes to Chilean fish-farming practices are vital for ensuring the continued productivity and well-being of farmed salmonids.

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Short-term storage of semen is a necessary key procedure in fish; it allows maximizing the use of gametes. Nevertheless, sperm quality decreases during storage has been associated with oxidative stress damage due to an increase in reactive oxygen species (ROS) during storage. This study was designed to optimize a short-term storage protocol for Coho salmon (Oncorhynchus kisutch) spermatozoa, evaluating the effect of extender dilution and the addition of butyl-hydroxytoluene (BHT) antioxidant on sperm function parameters. In the first experiment, fresh semen was diluted in Storfish®: extender dilution (1:2 and 1:3) and a control sample undiluted and stored at 4 °C for 7-days. In both experiments motility (MO), viability and integrity of plasma membrane, mitochondrial membrane potential (MMP) and superoxide anion level (O2-) were evaluated at 0, 3 and 7 days. Result shows that, 1:3 dilution maintained a higher sperm function for a longer period time. In the second experiment, spermatozoa were suspended in Storfish® (1:3) supplemented with two different concentrations of BHT (1.0 mM and 2.0 mM) and a control sample without antioxidant and stored at 4 °C for 7 days. The results demonstrated that, antioxidant-supplemented samples greater MO than control samples (P < 0.05). The viability remained >75% during storage in all groups. MMP was higher in 2.0 mM BHT compared to 1.0 mM and control (P < 0.05), in addition, this concentration reduced O2- level (P < 0.05). In conclusion, sperm: extender dilution 1:3 and adding of 2.0 mM BHT in sperm storage extender may enhance protection sperm function in Oncorhynchus kisutch against effects harmful of the oxidative stress during the in vitro storage.

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The characterization of runs of homozygosity (ROH), using high-density single nucleotide polymorphisms (SNPs) allows inferences to be made about the past demographic history of animal populations and the genomic ROH has become a common approach to characterize the inbreeding. We aimed to analyze and characterize ROH patterns and compare different genomic and pedigree-based methods to estimate the inbreeding coefficient in two pure lines (POP A and B) and one recently admixed line (POP C) of coho salmon (Oncorhynchus kisutch) breeding nuclei, genotyped using a 200 K Affymetrix Axiom® myDesign Custom SNP Array. A large number and greater mean length of ROH were found for the two "pure" lines and the recently admixed line (POP C) showed the lowest number and smaller mean length of ROH. The ROH analysis for different length classes suggests that all three coho salmon lines the genome is largely composed of a high number of short segments (<4 Mb), and for POP C no segment >16 Mb was found. A high variable number of ROH, mean length and inbreeding values across chromosomes; positively the consequence of artificial selection. Pedigree-based inbreeding values tended to underestimate genomic-based inbreeding levels, which in turn varied depending on the method used for estimation. The high positive correlations between different genomic-based inbreeding coefficients suggest that they are consistent and may be more accurate than pedigree-based methods, given that they capture information from past and more recent demographic events, even when there are no pedigree records available.

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