Salmo salar Skin and Gill Microbiome during Piscirickettsia salmonis Infection.

Maintaining the high overall health of farmed animals is a central tenant of their well-being and care. Intense animal crowding in aquaculture promotes animal morbidity especially in the absence of straightforward methods for monitoring their health. Here, we used bacterial 16S ribosomal RNA gene sequencing to measure bacterial population dynamics during P. salmonis infection. We observed a complex bacterial community consisting of a previously undescribed core pathobiome. Notably, we detected Aliivibrio wodanis and Tenacibaculum dicentrarchi on the skin ulcers of salmon infected with P. salmonis, while Vibrio spp. were enriched on infected gills. The prevalence of these co-occurring networks indicated that coinfection with other pathogens may enhance P. salmonis pathogenicity.

Genomics of Re-Emergent Aeromonas salmonicida in Atlantic Salmon Outbreaks.

Furunculosis, caused by Aeromonas salmonicida, poses a significant threat to both salmonid and non-salmonid fish in diverse aquatic environments. This study explores the genomic intricacies of re-emergent A. salmonicida outbreaks in Atlantic salmon (Salmo salar). Previous clinical cases have exhibited pathological characteristics, such as periorbital hemorrhages and gastrointestinal abnormalities. Genomic sequencing of three Chilean isolates (ASA04, ASA05, and CIBA_5017) and 25 previously described genomes determined the pan-genome, phylogenomics, insertion sequences, and restriction-modification systems. Unique gene families have contributed to an improved understanding of the psychrophilic and mesophilic clades, while phylogenomic analysis has been used to identify mesophilic and psychrophilic strains, thereby further differentiating between typical and atypical psychrophilic isolates. Diverse insertion sequences and restriction-modification patterns have highlighted genomic structural differences, and virulence factor predictions can emphasize exotoxin disparities, especially between psychrophilic and mesophilic strains. Thus, a novel plasmid was characterized which emphasized the role of plasmids in virulence and antibiotic resistance. The analysis of antibiotic resistance factors revealed resistance against various drug classes in Chilean strains. Overall, this study elucidates the genomic dynamics of re-emergent A. salmonicida and provides novel insights into their virulence, antibiotic resistance, and population structure.

Isolation of a New Infectious Pancreatic Necrosis Virus (IPNV) Variant from Genetically Resistant Farmed Atlantic Salmon (Salmo salar) during 2021-2022.

Infectious pancreatic necrosis (IPN), caused by IPNV, affects several species of farmed fish, particularly Atlantic salmon, and is responsible for significant economic losses in salmon aquaculture globally. Despite the introduction of genetically resistant farmed Atlantic salmon and vaccination strategies in the Chilean salmon industry since 2019, the number of IPN outbreaks has been increasing in farmed Atlantic salmon in the freshwater phase. This study examined gross and histopathological lesions of IPNV-affected fish, as well as the IPNV nucleotide sequence encoding the VP2 protein in clinical cases. The mortality reached 0.4% per day, and the cumulative mortality was from 0.4 to 3.5%. IPNV was isolated in the CHSE-214 cell line and was confirmed by RT-PCR, and VP2 sequence analysis. The analyzed viruses belong to IPNV genotype 5 and have 11 mutations in their VP2 protein. This is the first report of IPN outbreaks in farmed Atlantic salmon genetically resistant to IPNV in Chile. Similar outbreaks were previously reported in Scotland and Norway during 2018 and 2019, respectively. This study highlights the importance of maintaining a comprehensive surveillance program in conjunction with the use of farmed Atlantic salmon genetically resistant to IPNV.

Whole-Genome sequencing and comparative genomics of Mycobacterium spp. from farmed Atlantic and coho salmon in Chile.

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.

Extensive Phylogenetic Analysis of Piscine Orthoreovirus Genomic Sequences Shows the Robustness of Subgenotype Classification.

Piscine orthoreovirus (PRV) belongs to the family Reoviridae and has been described mainly in association with salmonid infections. The genome of PRV consists of about 23,600 bp, with 10 segments of double-stranded RNA, classified as small (S1 to S4), medium (M1, M2 and M3) and large (L1, L2 and L3); these range approximately from 1000 bp (segment S4) to 4000 bp (segment L1). How the genetic variation among PRV strains affects the virulence for salmonids is still poorly understood. The aim of this study was to describe the molecular phylogeny of PRV based on an extensive sequence analysis of the S1 and M2 segments of PRV available in the GenBank database to date (May 2020). The analysis was extended to include new PRV sequences for S1 and M2 segments. In addition, subgenotype classifications were assigned to previously published unclassified sequences. It was concluded that the phylogenetic trees are consistent with the original classification using the PRV genomic segment S1, which differentiates PRV into two major genotypes, I and II, and each of these into two subgenotypes, designated as Ia and Ib, and IIa and IIb, respectively. Moreover, some clusters of country- and host-specific PRV subgenotypes were observed in the subset of sequences used. This work strengthens the subgenotype classification of PRV based on the S1 segment and can be used to enhance research on the virulence of PRV.

Disentangling the environmental processes responsible for the world's largest farmed fish-killing harmful algal bloom: Chile, 2016.

The dictyochophyte microalga Pseudochattonella verruculosa was responsible for the largest farmed fish mortality ever recorded in the world, with losses for the Chilean salmon industry amounting to US$ 800 M in austral summer 2016. Super-scale climatic anomalies resulted in strong vertical water column stratification that stimulated development of a dynamic P. verruculosa thin layer (up to 38 µg chl a L-1) for several weeks in Reloncaví Sound. Hydrodynamic modeling (MIKE 3D) indicated that the Sound had extremely low flushing rates (between 121 and 200 days) in summer 2016. Reported algal cell densities of 7000-20,000 cells mL-1 generated respiratory distress in fish that was unlikely due to low dissolved oxygen (permanently >4 mg L-1). Histological examination of salmon showed that gills were the most affected organ with significant tissue damage and circulatory disorders. It is possible that some of this damage was due to a diatom bloom that preceded the Pseudochattonella event, thereby rendering the fish more susceptible to Pseudochattonella. No correlation between magnitude of fish mortality and algal cell abundance nor fish age was evident. Algal cultures revealed rapid growth rates and high cell densities (up to 600,000 cells mL-1), as well as highly complex life cycle stages that can be easily overlooked in monitoring programs. In cell-based bioassays, Chilean P. verruculosa was only toxic to the RTgill-W1 cell line following exposures to high cell densities of lysed cells (>100,000 cells mL-1). Fatty acid profiles of a cultured strain showed elevated concentrations of potentially ichthyotoxic, long-chain polyunsaturated fatty acids (PUFAs) (69.7% ± 1.8%)- stearidonic (SDA, 18:4ω3-28.9%), and docosahexaenoic acid (DHA, 22:6ω3-22.3%), suggesting that lipid peroxidation may help to explain the mortalities, though superoxide production by Pseudochattonella was low (< 0.21 ± 0.19 pmol O2- cell-1 h-1). It therefore remains unknown what the mechanisms of salmon mortality were during the Pseudochattonella bloom. Multiple mitigation strategies were used by salmon farmers during the event, with only delayed seeding of juvenile fish into the cages and towing of cages to sanctuary sites being effective. Airlift pumping, used effectively against other fish-killing HABs in the US and Canada was not effective, perhaps because it brought subsurface layers of Pseudochattonella to the surface, or and it also may have lysed the fragile cells, rendering them more lethal. The present study highlights knowledge gaps and inefficiency of contingency plans by the fish farming industry to overcome future fish-killing algal blooms under future climate change scenarios. The use of new technologies based on molecular methods for species detection, good farm practices by fish farms, and possible mitigation strategies are discussed.

Deformación de la aleta caudal en alevín de salmón del atlántico (Salmo salar), en etapa de saco vitelino / Deformation of the caudal fin in atlantic salmon fry (Salmo salar), in the yolk sac stage

RESUMEN: En este estudio se analiza una deformación que afectó la aleta caudal de los alevines de salmón del Atlántico (Salmo salar) y que les proporcionó un aspecto de "cola aguzada". Al momento de la eclosión se observaron completamente normales pero la deformación se detectó con posterioridad. El objetivo de este trabajo es describir los cambios anatómicos e histológicos de la aleta caudal deformada del alevín de salmón del Atlántico (Salmo salar). Para esto se utilizaron 60 alevines para realizar análisis de laboratorio y descartar la presencia de patógenos virales o bacterianos. Otros 60 alevines con un estado de desarrollo entre 600 y 700 Unidades térmicas acumuladas (UTAs) fueron anestesiados con Benzocaína 5 %, fijados en formalina al 10 % pesados y medidos. De estos un grupo de 30 alevines (15 normales y 15 deformes) fueron sometidos a la técnica de Hanken y Wassersug para evaluar Lepidotriquias. Los otros 30 alevines (15 normales y 15 deformes), fueron procesados mediante las técnicas histoquímicas: H&E/azul de Alcián para evaluar las características histológicas generales. Adicionalmente se utilizó técnicas inmunohistoquímicas para reconocer la ubicación y la presencia de los centros de señales Sonic hedgehog (Shh) para la formación de Lepidotriquias. A los valores obtenidos para las variables cuantitativas peso y longitud de cuerpo, largo y ancho de aleta caudal, se les realizó estadística descriptiva y fueron sometidos a prueba de normalidad de Shapiro-Wilk. Las diferencias observadas entre peces normales y deformes, fueron analizadas mediante prueba t de Student o U de Mann Whitney, utilizando el paquete estadístico IBM SPSS 20.0. La deformación se observó desde las 600 UTA. El peso de los alevines deformes fue similar al de los peces normales (p>0,05), lo mismo sucedió con la longitud de la aleta (p>0,05). Por el contrario, el ancho de la aleta de los deformes fue muy reducida (p<0,05). El fenotipo de aleta aguzada presentó un cambio en la integridad de los bordes, fracturas de Lepidotriquias. La epidermis de la aleta caudal de los alevines deformados presentó sus centros de señalización Shh activos, pero el blastema interrayos presentó vasodilatación, congestión y hemorragias. La presentación de este caso se relacionó con incrementos bruscos de temperatura peri eclosional.

SUMMARY: This study analyzes the deformity of Atlantic salmon fry (Salmo salar) caudal fin, which gives it a "pointed tail" appearance. Although at hatching specimens were normal, the deformities were detected later. The objective of this work is to describe the anatomical and histological changes of the deformed caudal fin of the Atlantic salmon fry (Salmo salar). In this analysis we used 60 specimens for laboratory analyses, to rule out the presence of viral or bacterial pathogens. Another 60 fry, developmental stage between 600 and 700 Accumulated Thermal Units (UTAs) were anesthetized with 5 % Benzocaine, fixed in 10 % formalin, weighed and measured. Of these, a group of 30 fry (15 normal and 15 deformed) underwent the Hanken and Wassersug technique to evaluate lepidotrychia or dermal rays. The remaining 30 fry (15 normal and 15 deformed) were processed using H & E / Alcián blue histochemical techniques to evaluate general histological characteristics. Additionally, immunohistochemical techniques were used to determine the location and presence of Sonic hedgehog (Shh) signal centers for lepidotrychia development. The values obtained for the quantitative variables body weight, length and width of the tail fin were described using the arithmetic mean and standard deviation. The deformity was observed from 600 UTA. Weight of deformed fry was less than normal fish, length of the fin was similar in normal and deformed fish. In contrast, width of the deformed fin was significantly reduced. The sharp fin phenotype presented a change in the conformity of the edges, lepidotrychia fractures. The epidermis presented active Shh signaling centers, but the interray blastema showed vasodilation, congestion and hemorrhages. The presentation of this case was related to sudden increases in perieclosional temperature.

Microbial Diversity of the Red Sea Urchin Loxechinus albus during Controlled Farming in Puerto Montt, Chile, Using 16S rRNA Gene Amplicon Sequencing.

Loxechinus albus is a shallow-water sea urchin, and its distribution is related to the cold water of the Southern Hemisphere. Recently, bacterial communities, also called microbiota, in sea urchins have started being explored. In this report, we have characterized the surface, testa, and gonad microbiota using 16S rRNA sequencing.

Genomic epidemiology of the commercially important pathogen Renibacterium salmoninarum within the Chilean salmon industry.

Renibacterium salmoninarum is the causative agent of bacterial kidney disease (BKD), which is a commercially important disease of farmed salmonids. Typing by conventional methods provides limited information on the evolution and spread of this pathogen, as there is a low level of standing variation within the R. salmoninarum population. Here, we apply whole-genome sequencing to 42 R. salmoninarum isolates from Chile, primarily from salmon farms, in order to understand the epidemiology of BKD in this country. The patterns of genomic variation are consistent with multiple introductions to Chile, followed by rapid dissemination over a 30 year period. The estimated dates of introduction broadly coincide with major events in the development of the Chilean aquaculture industry. We find evidence for significant barriers to transmission of BKD in the Chilean salmon production chain that may also be explained by previously undescribed signals of host tropism in R. salmoninarum. Understanding the genomic epidemiology of BKD can inform disease intervention and improve sustainability of the economically important salmon industry. This article contains data hosted by Microreact.

First description of clinical presentation of piscine orthoreovirus (PRV) infections in salmonid aquaculture in Chile and identification of a second genotype (Genotype II) of PRV.

BACKGROUND: Heart and skeletal muscle inflammation (HSMI) is an emerging disease of marine-farmed Atlantic salmon Salmo salar, first recognized in 1999 in Norway, and recently associated with piscine orthoreovirus (PRV) infection. To date, HSMI lesions with presence of PRV have only been described in marine-farmed Atlantic salmon in Norway. A new HSMI-like disease in rainbow trout Oncorhynchus mykiss associated with a PRV-related virus has also been reported in Norway. METHODS: Sampling of Atlantic salmon and coho salmon was done during potential disease outbreaks, targeting lethargic/moribund fish. Fish were necropsied and tissues were taken for histopathologic analysis and testing for PRV by RT-qPCR assay for segment L1 and conventional RT-PCR for PRV segment S1. The PCR products were sequenced and their relationship to PRV strains in GenBank was determined using phylogenetic analysis and nucleotide and amino acid homology comparisons. RESULTS: The Atlantic salmon manifested the classical presentation of HSMI with high PRV virus loads (low Ct values) as described in Norway. The coho salmon with low Ct values had myocarditis but only in the spongy layer, the myositis of red muscle in general was mild, and the hepatic necrosis was severe. Upon phylogenetic analysis of PRV segment S1 sequences, all the Chilean PRV strains from Atlantic salmon grouped as sub-genotype Ib, whereas the Chilean PRV strains from coho salmon were more diversified, grouping in both sub-genotypes Ia and Ib and others forming a distinct new phylogenetic cluster, designated Genotype II that included the Norwegian PRV-related virus. CONCLUSIONS: To our knowledge the present work constitutes the first published report of HSMI lesions with presence of PRV in farmed Atlantic salmon outside of Europe, and the first report of HSMI-like lesions with presence of PRV in coho salmon in Chile. The Chilean PRV strains from coho salmon are more genetically diversified than those from Atlantic salmon, and some form a distinct new phylogenetic cluster, designated Genotype II.

Genetic analysis and comparative virulence of infectious salmon anemia virus (ISAV) types HPR7a and HPR7b from recent field outbreaks in Chile.

BACKGROUND: Infectious salmon anemia (ISA) is a serious disease of marine farmed Atlantic salmon, Salmo salar L. caused by ISA virus (ISAV). ISAV genomic segments 5 and 6 encode surface glycoproteins hemagglutinin-esterase (HE) and F protein important for the pathogenicity of ISAV. In this study, we describe the genetic characteristics and relationship between ISAV-HPR7a and ISAV-HPR7b strains that caused the ISA outbreaks in Chile in 2013 and 2014, respectively, and the evolution of the ISAV clades since 2009 based on segment 5 and 6 sequences. METHODS: The study material included samples from six ISA cases in Chile. RNA was extracted from salmon tissues and ISAV isolated from cell culture; segments 5 and 6 were amplified by RT-PCR and compared by alignment with ISAV sequences from the GenBank database. RESULTS: ISAV-HPR7a and ISAV-HPR7b belong to the European Genotype I strains only found in Europe and Chile, and in both cases, show high similarity in segments 5 and 6 with identity between 95-96%. Our data confirm the hypothesis that the original virus was introduced to Chile in 1996. Compared to the 2007 ISAV-HPR7b isolate, the 2014 ISAV-HPR7b does not have an insertion in segment 5 and was associated with low mortality, which suggests that ISAV virulence was attenuated by the absence of the insertion in segment 5. In contrast, the highly virulent ISAV-HPR14 from April 2013 outbreak did not have the insertion in segment 5 either. CONCLUSION: Variability in the ISAV virulence markers supports the quasispecies theory that multiple evolution forces are likely to shape ISAV genetic diversity. Our findings provide evidence of continuing evolution of ISAV in the Chilean aquaculture industry.

Genome Sequence of Streptococcus phocae subsp. salmonis Strain C-4T, Isolated from Atlantic Salmon (Salmo salar).

Streptococcus phocae subsp. salmonis is a fish pathogen that has an important impact on the Chilean salmon industry. Here, we report the genome sequence of the type strain C-4(T) isolated from Atlantic salmon (Salmo salar), showing a number of interesting features and genes related to its possible virulence factors.

Infectious salmon anaemia virus (ISAV) in Chilean Atlantic salmon (Salmo salar) aquaculture: emergence of low pathogenic ISAV-HPR0 and re-emergence of virulent ISAV-HPR∆: HPR3 and HPR14.

ABSTACT: Infectious salmon anaemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), which belongs to the genus Isavirus, family Orthomyxoviridae. ISA is caused by virulent ISAV strains with deletions in a highly polymorphic region (HPR) of the hemagglutinin-esterase (HE) protein (designated virulent ISAV-HPR∆). This study shows the historic dynamics of ISAV-HPR∆ and ISAV-HPR0 in Chile, the genetic relationship among ISAV-HPR0 reported worldwide and between ISAV-HPR0 and ISAV-HPR∆ in Chile, and reports the 2013 ISA outbreak in Chile. The first ISA outbreak in Chile occurred from mid-June 2007 to 2010 and involved the virulent ISAV-HPR7b, which was then replaced by a low pathogenic ISAV-HPR0 variant. We analyzed this variant in 66 laboratory-confirmed ISAV-HPR0 cases in Chile in comparison to virulent ISAV-HPR∆ that caused two new ISA outbreaks in April 2013. Multiple alignment and phylogenetic analysis of HE sequences from all ISAV-HPR0 viruses allowed us to identify three genomic clusters, which correlated with three residue patterns of ISAV-HPR0 (360PST362, 360PAN362 and 360PAT362) in HPR. The virus responsible for the 2013 ISAV-HPR∆ cases in Chile belonged to ISAV-HPR3 and ISAV-HPR14, and in phylogenetic analyses, both clustered with the ISAV-HPR0 found in Chile. The ISAV-HPR14 had the ISAV-HPR0 residue pattern 360PAT362, which is the only type of ISAV-HPR0 variant found in Chile. This suggested to us that the 2013 ISAV-HPR∆ re-emerged from ISAV-HPR0 that is enzootic in Chilean salmon aquaculture and were not new introductions of virulent ISAV-HPR∆ to Chile. The clinical presentations and diagnostic evidence of the 2013 ISA cases indicated a mixed infection of ISAV with the ectoparasite Caligus rogercresseyi and the bacterium Piscirickettsia salmonis, which underscores the need for active ISAV surveillance in areas where ISAV-HPR0 is enzootic, to ensure early detection and control of new ISA outbreaks, as it is considered a risk factor. This is the first report of ISA linked directly to the presence of ISAV-HPR0, and provides strong evidence supporting the contention that ISAV-HPR0 shows a strong relationship to virulent ISAV-HPR∆ viruses and the possibility that it could mutate to virulent ISAV-HPR∆.