Diseased Rainbow Trout Research Articles

Highly Efficient Methods with a Generalized Linear Mixed Model for the Quantitative Trait Locus Mapping of Resistance to Columnaris Disease in Rainbow Trout (Oncorhynchus mykiss)

Linear mixed models (LMMs) are commonly used in genome-wide association studies (GWASs) to evaluate population structures and relatedness. However, LMMs have been shown to be ineffective in controlling false positive errors for the analysis of resistance to Columnaris disease in Rainbow Trout. To solve this problem, we conducted a series of studies using generalized linear mixed-model association software such as GMMAT (v1.4.0) (generalized linear mixed-model association tests), SAIGE (v1.4.0) (Scalable and Accurate Implementation of Generalized mixed model), and Optim-GRAMMAR for scanning a total of 25,853 SNPs. Seven different SNPs (single-nucleotide polymorphisms) associated with the trait of resistance to Columnaris were detected by Optim-GRAMMAR, four SNPs were detected by GMMAT, and three SNPs were detected by SAIGE, and all of these SNPs can explain 8.87% of the genetic variance of the trait of resistance to Columnaris disease. The heritability of the trait of resistance to Columnaris re-evaluated by GMMAT was calibrated and was found to amount to a total of 0.71 other than 0.35, which was seriously underestimated in previous research. The identification of LOC110520307, LOC110520314, and LOC110520317 associated with the resistance to Columnaris disease will provide us more genes to improve the genetic breeding by molecular markers. Finally, we continued the haplotype and gene-based analysis and successfully identified some haplotypes and a gene (TTf-2) associated with resistance to Columnaris disease.

Isolation, Identification, and Molecular Evolutionary Analysis of Infectious Hematopoietic Necrosis Virus (IHNV) from Farmed Rainbow Trout in Southwest China

IHNV troubled the salmon breeding industry in China for years, and it often causes large-scale deaths. To study the disease characteristics of IHNV and its epidemiology in China， this study isolated and sequenced the Whole genome of an IHNV strain (IHNV-SCCD) (GenBank No： OQ801357) from farmed diseased rainbow trout in Chengdu and compared 99 IHNV strains from China by mega.11. The IHNV-SCCD strain was isolated by using EPC cells. The complete genome sequence was determined and deposited in the NCBI database. Phylogenetic analysis showed that IHNV-SCCD belongs to the typical J gene group, recently with Sichuan strains. Except for BjLL strain and LN12-17 strain, the remaining 98 strains followed a single evolutionary path. Note that IHNV in Liaoning province is the fastest evolutionary. In addition to the geographical differences, the Xinjiang strains were closely related to the Yunnan strains, and some strains from Beijing were closely related to some strains from Sichuan. There were two evolutionary branches in Gansu and Qinghai; QH17, isolated from Qinghai, was most closely related to SX1704 from Shaanxi. These results suggest that the prevalence of IHNV in China was extremely wide, and the evolution rate was fast. It is necessary to strengthen the monitoring and prevention methods.

The impact of apple cider vinegar on nonalcoholic fatty liver disease in rainbow trout, Oncorhynchus mykiss: A study of therapeutic potential and health benefits

AbstractIn this study, we aimed to experimentally induce fatty liver disease in rainbow trout, Oncorhynchus mykiss, and then assessed the illness recovery process, growth, and changes in the expression of FAAH and ACADL genes in both healthy (0 [C2] and 4% apple cider vinegar [T4]) and diseased fish (0 [C1], 1 [T1], 2 [T2], and 4% [T3]) apple cider vinegar. To conduct the study, 180 rainbow trout were randomly assigned to six different experimental treatments, each with three replications. The investigation lasted for 60 days. Growth indices, liver histology, blood biochemical parameters, and transcription of the ACADL and FAAH genes in the liver tissue were measured. The study found no significant differences in the final weights across all the treatments. Apple cider vinegar (ACV) administration resulted in a decrease in AST, ALT, and ALP; however, these values did not show a significant difference from C2. In T3, triglycerides significantly decreased (p < 0.05), whereas in T4, triglycerides significantly increased (p < 0.05). Hepatocytes from ACV‐containing treatments showed reduced fat compared with T4 and the control group (C1). While there was a significant difference (p < 0.05) in the expression of the FAAH gene, there was no significant difference (p > 0.05) in the expression of the ACADL gene between experimental treatments. The findings of our study indicate that an inclusion of up to 2% ACV may have positive effects on trout aquaculture and NAFLD treatment.

Investigation of vibriosis caused by Vibrio anguillarum in rainbow trout (Oncorhynchus mykiss).

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 (Muğla-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.

Characterization of Tenacibaculum maritimum isolated from diseased salmonids farmed in Chile reveals high serological and genetic heterogeneity.

The diversity of Tenacibaculum maritimum in Chile remains poorly understood, particularly in terms of antigenic and genetic diversity. This information is crucial for the future development of a vaccine against tenacibaculosis and would increase understanding of this important fish pathogen. With this aim, the biochemical, antigenic, and genetic characteristics were analysed for 14 T. maritimum isolates, recovered from diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) farmed in Chile between 1998 and 2022. Biochemical analysis showed a homogeneity among all the Chilean T. maritimum isolates and all four other strains included for comparison purposes. Serological characterization using dot-blot assaying revealed antigenic heterogeneity with the use of unabsorbed antisera. The majority of isolates showed cross-reactions, identifying three main serological patterns. When the PCR-based serotyping scheme was performed, the existence of antigenic heterogeneity was confirmed. Four Atlantic salmon isolates were 4-0; and most isolates, including the rainbow trout isolate, were 3-1 (n = 9). A turbot (Scophthalmus maximus) isolate was 1-0. Using an existing Multilocus Sequence Typing system, two newly identified sequence types (ST193 and ST198) in the database were detected. ST193 encompassed nine isolates obtained from Atlantic salmon and rainbow trout, while ST198 regrouped four isolates, all retrieved from diseased Atlantic salmon in 2022. These findings highlight significant antigenic and genetic diversity among the Chilean isolates. This information is useful for epizootiology and the selection of suitable candidate strain(s) for vaccine development against tenacibaculosis caused by T. maritimum in Chilean salmon farming.

Viral Diseases in Trout and Treatment Methods

Viral diseases cause significant economic losses in aquaculture enterprises in Turkey and around the world. In this review, information is given about the symptoms, diagnosis and treatment of infectious pancreatic necrosis (IPN), infectious hematopoietic necrosis (IHN), Viral hemorrhagic septicemia (VHS) diseases in rainbow trout (Oncorhynchus mykis) in different periods.

Novel insights on microbiome dynamics during a gill disease outbreak in farmed rainbow trout (Oncorhynchus mykiss)

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.

Use of tuf gene sequencing for Identification of Lactococci isolated from diseased rainbow trout (Oncorhynchus mykiss)

Lactococcosis is a bacterial infection that is characterized by the appearance of septicemia in fish. Traditionally, the identification of causes of disease outbreaks in aquaculture poses a major challenge due to their comparable characteristics. In this study, simple PCR and sequencing based on the tuf gene were developed to identify isolates belonging to the Streptococcaceae family. The outbreaks of lactococcosis in rainbow trout have been mainly associated with Lactococcus garvieae, but the emergence of closely related species, Lactococcus petauri and Lactococcus formosensis pose a challenge in accurate identification. The study aimed to identify Gram-positive catalase-negative cocci and differentiate between Lactococcosis-causing bacteria (LCB) in rainbow trout isolates associated with lactococcosis outbreaks in Iran. Seventy-five Gram-positive cocci strains from diseased rainbow trout were tested using a PCR and sequencing method targeting the tuf gene. Sequence and phylogenetic analyses were conducted to distinguish between closely related Lactococcus species. The PCR results revealed a successful amplification with tuf gene primers for all strains, allowing species identification. The sequence analysis showed that all 29 isolates initially identified as L.garvieae were 100% identical to L.petauri, indicating a previous misclassification. This work is the first report by L.petauri of Iran. Analysis of tuf gene sequences revealed different patterns of single nucleotide polymorphism (SNP) for accurate species differentiation and phylogenetic analysis confirmed different clusters for LCB. The study highlights the misidentification of L.petauri as L.garvieae in lactococcosis outbreaks and the importance of accurate species differentiation in disease management. The tuf gene sequencing method shows promise for practical, cost-effective, and accurate differentiation of closely related streptococcal species in aquaculture. These findings strongly indicate the need to re-evaluate the initial identification of isolates from fish with lactococcisis. The findings of this study also suggest that the tuf gene sequencing, established in this research for its practicality and cost-efficiency, could potentially be integrated into routine diagnostic procedures for Gram-positive, catalase-negative cocci associated with outbreaks of infections in aquatic animals; however, further experimental works are warranted.

Characterisation of sRNAs enriched in outer membrane vesicles of pathogenic Flavobacterium psychrophilum causing Bacterial Cold Water Disease in rainbow trout.

Flavobacterium psychrophilum (Fp) causes Bacterial Cold Water Disease in salmonids. During host-pathogen interactions, gram-negative bacteria, such as Fp, release external membrane vesicles (OMVs) harbouring cargos, such as DNA, RNA and virulence factors. This study aimed to characterise the potential role of the OMVs' small RNAs (sRNAs) in the Fp-rainbow trout host-pathogen interactions. sRNAs carried within OMVs were isolated from Fp. RNA-Seq datasets from whole-cell Fp and their isolated OMVs indicated substantial enrichment of specific sRNAs in the OMVs compared to the parent cell. Many of the OMV-packaged sRNAs were located in the pathogenicity islands of Fp. Conservation of sRNAs in 65 strains with variable degrees of virulence was reported. Dual RNA-Seq of host and pathogen transcriptomes on day 5 post-infection of Fp -resistant and -susceptible rainbow trout genetic lines revealed correlated expression of OMV-packaged sRNAs and their predicted host's immune gene targets. In vitro, treatment of the rainbow trout epithelial cell line RTgill-W1 with OMVs showed signs of cytotoxicity accompanied by dynamic changes in the expression of host genes when profiled 24 h following treatment. The OMV-treated cells, similar to the Fp -resistant fish, showed downregulated expression of the suppressor of cytokine signalling 1 (SOCS1) gene, suggesting induction of phagosomal maturation. Other signs of modulating the host gene expression following OMV-treatment include favouring elements from the phagocytic, endocytic and antigen presentation pathways in addition to HSP70, HSP90 and cochaperone proteins, which provide evidence for a potential role of OMVs in boosting the host immune response. In conclusion, the study identified novel microbial targets and inherent characteristics of OMVs that could open up new avenues of treatment and prevention of fish infections.

Complete genome sequence and potential pathogenic assessment of Flavobacterium plurextorum RSG-18 isolated from the gut of Schlegel's black rockfish, Sebastes schlegelii.

Flavobacterium plurextorum is a potential fish pathogen of interest, previously isolated from diseased rainbow trout (Oncorhynchus mykiss) and oomycete-infected chum salmon (Oncorhynchus keta) eggs. We report here the first complete genome sequence of F. plurextorum RSG-18 isolated from the gut of Schlegel's black rockfish (Sebastes schlegelii). The genome of RSG-18 consists of a circular chromosome of 5,610,911 bp with a 33.57% GC content, containing 4858 protein-coding genes, 18 rRNAs, 63 tRNAs and 1 tmRNA. A comparative analysis was conducted on 11 Flavobacterium species previously reported as pathogens or isolated from diseased fish to confirm the potential pathogenicity of RSG-18. In the SEED classification, RSG-18 was found to have 36 genes categorized in 'Virulence, Disease and Defense'. Across all Flavobacterium species, a total of 16 antibiotic resistance genes and 61 putative virulence factors were identified. All species had at least one phage region and type I, III and IX secretion systems. In pan-genomic analysis, core genes consist of genes linked to phages, integrases and matrix-tolerated elements associated with pathology. The complete genome sequence of F. plurextorum RSG-18 will serve as a foundation for future research, enhancing our understanding of Flavobacterium pathogenicity in fish and contributing to the development of effective prevention strategies.

Traditional Chinese medicine bufalin inhibits infectious hematopoietic necrosis virus infection in vitro and in vivo.

Infectious hematopoietic necrosis virus (IHNV) is the pathogen of infectious hematopoietic necrosis (IHN) which outbreak often causes huge economic losses and hampers the healthy development of salmon and trout farming. Currently, there is only one approved DNA vaccine for IHN worldwide, but it faces some biosafety problems. Hence, more effective vaccines and antiviral drugs are needed to prevent IHNV infection. In this study, we report that bufalin, a traditional Chinese medicine, shows potential antiviral activity against IHNV both in vitro and in vivo. The bufalin-mediated block of IHNV infection occurred at the viral attachment and RNA replication stages, but not internalization, and bufalin inhibited IHNV infection by targeting Na+/K+-ATPase. The in vitro and in vivo studies showed that bufalin significantly inhibited IHNV infection and may be a promising candidate drug against the disease in rainbow trout.

Immersion challenge of three salmonid species (family Salmonidae) with three multilocus sequence typing variants of Flavobacterium psychrophilum provides evidence of differential host specificity.

Bacterial coldwater disease (BCWD), caused by Flavobacterium psychrophilum, results in significant losses among multiple salmonid (family Salmonidae) species. Molecular epidemiology and serotyping studies have suggested that some variants are host specific; however, these associations have not been evaluated by cross-challenging fish species with putatively host-associated F. psychrophilum isolates via more natural (i.e. immersion) exposure routes. To this end, F. psychrophilum isolates US19-COS, US62-ATS and US87-RBT, each originally recovered from diseased coho salmon (Oncorhynchus kisutch), Atlantic salmon (Salmo salar) or rainbow trout (O. mykiss), and belonging to a host-associated multilocus sequence typing clonal complex (e.g. CC-ST9, CC-ST232 or CC-ST10), were PCR-serotyped, evaluated for proteolytic activity, and used to challenge adipose fin-clipped 4-month old Atlantic salmon, coho salmon and rainbow trout via immersion. Findings showed US87-RBT caused disease and mortality only in rainbow trout (e.g. 56.7% survival probability). US19-COS and US62-ATS caused more mortality in coho salmon and Atlantic salmon but also caused disease in both other host species, albeit to a lesser extent. Observed survival differences may be due to variant antigenic/virulence determinants as differences in serotype and proteolytic activity were discovered. Collectively, results highlight the intricacies of F. psychrophilum-host interactions and provide further invivo evidence that some F. psychrophilum MLST variants are host specific, which may have implications for the development of BCWD prevention and control strategies.

The antiviral effects of TRIM23 and TRIM32 proteins in rainbow trout (Oncorhynchus mykiss)

TRIM proteins play a crucial antiviral effector role in the innate immune system of vertebrates. In this study, we found that TRIM proteins exhibited the highest expression levels in immune organs such as spleen and kidney during IHNV infection in rainbow trout, meanwhile, we successfully amplified TRIM23 and TRIM32 from diseased rainbow trout and analyzed their gene sequences, revealing that rainbow trout TRIM23 and TRIM32 proteins are closely related to Atlantic salmon and Chinook salmon; In this experiment, the TRIM23 and TRIM32 protein genes were resoundingly constructed as a recombinant plasmids and expressed in CHSE-214 cells. Upon transfected with the recombinant plasmid, followed by viral infection, significant decreasion in the copy numbers of the virus was observed, indicating that the TRIM23 and TRIM32 proteins of rainbow trout play an important role in inhibiting virus replication, with the TRIM32 role being the most pronounced. These results provide a basis for subsequent in-depth study of the antiviral effects of TRIM proteins, and provide new ideas for immune enhancers.

Characterization and Pathogenicity of Flavobacterium psychrophilum Isolated from Rainbow Trout (Oncorhynchus mykiss) in Korea.

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease in salmonids and rainbow trout fry syndrome. This pathogen has attained a global presence and can spread both horizontally and vertically. However, it was not documented in Korea before September 2018. In this study, the objectives were to characterize Flavobacterium psychrophilum strain FPRT1, isolated from diseased rainbow trout genotypically and phenotypically. We also conducted various investigations to better understand its impact and assess potential control measures. We acquired fifty rainbow trout (approximately 70 g in weight) and transferred them to a laboratory aquarium. During the initial acclimation period, we observed mortality and examined affected fish for clinical signs. We isolated the bacterium from the spleen of infected rainbow trout using tryptone yeast extract salts agar supplemented with glucose, naming this FPRT1. Antibiotic susceptibility testing was carried out, and from the result, we selected enrofloxacin to administer to the trout orally to reduce mortality. To evaluate pathogenicity, we exposed the trout to FPRT1 at different water temperatures (8, 15, and 22 °C). Genomic analysis was conducted to identify the serotype and relatedness of FPRT1 to European strains. Affected fish displayed clinical signs, such as ulcerative lesions in the mandible, anemia with pale gills, exophthalmia, and increased mucus secretion. Internal symptoms included pale liver and enlarged spleen. FPRT1 was susceptible to erythromycin, enrofloxacin, florfenicol, oxytetracycline, and gentamicin, but resistant to oxolinic acid and sulfamethoxazole/trimethoprim. Oral administration of enrofloxacin resulted in a decrease in mortality from 28% to 6%. Pathogenicity tests revealed varying mortality rates due to FPRT1 at different temperatures. The highest rates were observed at 8 °C (ranging from 43% to 100%) for both intraperitoneal and intramuscular injections, and lower rates occurred at 22 °C (ranging from 0% to 30%), with intramuscular injections displaying higher susceptibility. Genomic analysis identified FPRT1 as serotype 2 and indicated its close genetic relationship with European strains based on the core genome and dispensable genome. The substantial genomic similarity between our strain and European strains suggests the possibility of bacterial spread through the importation of fertilized eggs from Europe. In conclusion, this study highlights the introduction of the previously undocumented pathogen (F. psychrophilum) into Korean rainbow trout populations. The detection of this pathogen and its pathogenicity assessment is not only important for understanding its impact on local aquaculture but also for establishing surveillance and control measures to prevent further transmission and outbreaks in the region.

Pathological analysis and antimicrobial susceptibility of Chryseobacterium balustinum RTFCP 298 isolated from diseased rainbow trout, Oncorhynchus mykiss

In this study, six isolates of Chryseobacterium balustinum were characterized from diseased rainbow trout fingerlings. The virulence characteristics, pathogenicity, and antimicrobial susceptibility pattern of these isolates were investigated. The bacterium showed positive results for catalase, cytochrome oxidase, and aesculin hydrolysis, while negative results were obtained for DNase, gelatinase, methyl red, Voges-Proskauer's reaction, Simon citrate, Hydrogen sulphide, and starch hydrolysis. Amino acid metabolism analysis revealed the inability to metabolize arginine, lysine, and ornithine decarboxylase. Molecular characterization (16S rRNA) and phylogenetic analysis revealed the test isolates as C. balustinum, closely related to strain WLT (99.85% similarity) and C. balustinum P-27 (99.77%). Virulence assay indicated haemolytic activity and biofilm formation by the test bacterium. The challenge test confirmed moderate pathogenicity in rainbow trout and established Koch's postulates. The clinical manifestations of infection included fin erosion, eye and body surface haemorrhage, exophthalmia, and organ liquefaction. The minimum inhibitory concentrations of various antimicrobials ranged from 1 to > 256 µg mL−1. The novel synthetic antimicrobial peptides exhibited MICs of 8 to > 256 µg mL−1, suggesting a potential control method. These findings suggest that C. balustinum is an opportunistic pathogen with moderate pathogenicity in rainbow trout. Further research on the host–pathogen relationship is necessary to understand virulence characteristics and pathogenicity in aquaculture.

Co-occurrence of heterogeneous Flavobacterium psychrophilum isolates within the same Chilean farm and during the same infectious outbreak.

Flavobacterium psychrophilum is a pathogenic bacterium affecting Chilean salmonid farms. High antigenic and genetic diversity exists among Chilean F. psychrophilum isolates, but the distribution thereof among farms is poorly understood. These epidemiological data are key for developing isolate-specific vaccines. The present study isolated F. psychrophilum in diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) from five freshwater farms between 2018 and 2019. Each farm only raised one salmonid species and was geographically separated from and did not share culturing water with the other farms. Antigenic and genetic analyses were conducted to shed light on the possibility of isolates coexisting within the same farm during outbreaks. A total of 68 Chilean F. psychrophilum isolates were recovered from skin lesions, gills, fins, kidney and spleen of moribund and live fish. Among the 68 Chilean isolates, mPCR-serotyping indicated three major serotypes (i.e. 23.5% type 0; 47.1% type 2; and 26.5% type 4) and, to a lesser degree, serotype 1 (2.9%). Sixteen antigenic groups were detected by slide agglutination. Genetic characterizations by 16S rRNA alleles identified 71% of the isolates as the virulent genogroup CSF259-93 allele. A predominant serotype was associated with each farm, with types 0 and 4 related to Atlantic salmon and types 1 and 2 to rainbow trout. Notwithstanding, several antigenic groups coexisted within some farms. Likewise, the experimental intramuscular challenges (n = 20) demonstrated that the type-2 isolates from rainbow trout were the most pathogenic among isolates recovered from infectious outbreaks in Atlantic salmon, especially as compared to those from types-0 and -4. These results allow us to suggest that prevention measures, specifically vaccines, should be developed according to dominant isolates and with specificity to each farm, that is the use of autogenous or site-specific vaccines.

ТHE SIGNIFICANCE OF CERULOPLASMIN AND C-REACTIVE PROTEIN AS POSSIBLE MARKERS FOR THE DIAGNOSIS OF MYXOBOLOUS INFECTION CAUSED BY Myxobolus cerebralis IN RAINBOW TROUT (Oncorhynchus mykiss)

Myxobolus cerebralis is the etiological agent of one of the most devastating diseases (Whirling disease) in salmonids. It belongs to subphylum Myxozoa which possesses the highest susceptibility to the rainbow trout (Oncorhynchus mykiss). Acute phase proteins as an essential component of normal homeostasis are well defined and are presently used as a diagnostic and prognostic marker in human and animal pathology for evaluation of infection and inflammation. In piscine medicine the importance and the function of acute phase proteins still has not been fully determined. Furthermore, parasitic infections are more difficult to diagnose than bacterial and viral infections due to the lack of specific diagnostic tools therefore the change in the levels of some acute phase proteins could possibly be used as an alternative marker for myxobolosis. Further research is still needed to develop an understanding of the diagnostic significance of acute phase proteins in fish and the differentiation of their values in different types of disorders. The present study evaluated the modulation of ceruloplasmin and C-reactive protein following inflammatory stimulus by natural infection of Myxobolus cerebralis in rainbow trout (Oncorhynchus mykiss). In the research, biochemical tests and enzyme-linked immunosorbent assay were carried out. The test material was a diseased rainbow trout with a mortality rate of 45%. In the future, acute phase proteins have a real potential to become widely used as diagnostic tools in piscine medicine, as the evaluation of their levels can help better diagnosis and treatment, especially when it occurs during illness. Measurement of C-reactive protein concentration in trout may be used useful as a bioindicator of the health condition of this species. Further research is still needed to develop an understanding of the diagnostic significance of acute phase proteins in fish and the differentiation of their values in different types of disorders.

Effect of Different Levels of Chlorogenic Acid on Growth Performance, Immunological Responses, Antioxidant Defense, and Disease Resistance of Rainbow Trout (Oncorhynchus mykiss) Juveniles.

The current study is designed to assay the efficacy of chlorogenic acid (ChA) in the diet on growth performance, digestive enzyme activity, serum immunological, biochemical, and antioxidant variables, and mucosal immune response as well as disease resistance of rainbow trout (Oncorhynchus mykiss) juveniles. Rainbow trout juveniles received diets supplemented with different inclusion levels of ChA (0 (ctrl), 200 (CA1), 400 (CA2), 600 (CA3), and 800 (CA4) mg kg-1 diet) for 60 days. According to the findings, fish from CA3 and CA4 groups demonstrated the best results considering the final weight (FW) and weight gain (WG) (P < 0.05). Also, the group that received 600 mg kg-1 ChA-supplemented diet showed the lowest feed conversion ratio (FCR) and the highest specific growth rate (SGR) compared to other groups (P < 0.05). Moreover, the minimum survival rate (SR) was only detected in the CA4 treatment (P < 0.05). Regression analysis exhibited that rainbow trout growth indices were polynomially linked to dietary chlorogenic acid concentrations. In this regard, the optimal levels of chlorogenic acid according to growth parameters (FCR and SGR) were 0.71 and 0.62 gr kg-1 diet, respectively. The results exhibited superior performance of protease and amylase activities in CA2, CA3, and CA4 groups with the maximum amount in the group receiving 600 mg kg-1 ChA-enriched diet (P < 0.05). Serum lysozyme (LYZ), immunoglobulin (Ig), and components 3 and 4 (C3 and C4) values of CA2, CA3, and CA4 groups were significantly higher than others with the highest amount in the CA3 group (P <0.05). Additionally, serum nitroblue tetrazolium (NBT) value in the CA3 and CA4 groups and myeloperoxidase (MPO) in the CA3 group were notably more than others (P < 0.05). Moreover, the lowest aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) and the highest total protein (TP) and globulin (GLO) values were observed in CA3 treatment (P < 0.05). CA2 and CA3 groups demonstrated increased serum catalase (CAT) and decreased malondialdehyde (MDA) values compared to the control while the highest CAT and lowest MDA values were observed in CA3 treatment (P < 0.05). Considering mucus immunity, the significantly maximum LYZ and protease values were demonstrated in CA2 and CA3 groups, and the highest ALP, Ig, and esterase values were demonstrated in the CA3 group. In comparison with the control, the mortality rates of the groups that received the ChA diets were remarkably (P < 0.05) lower postchallenge with Y. ruckeri, and the highest survival and relative percentage of survival (RPS) (P < 0.05) belonged to the CA3 group. Results obtained from the current study suggested ChA as a functional dietary additive to raise growth parameters, immune indices, antioxidant capacity, and resistance to disease in rainbow trout.

Description of a Novel Fish Pathogen, Plesiomonas shigelloides subsp. oncorhynchi, Isolated from Rainbow Trout (Oncorhynchus mykiss): First Genome Analysis and Comparative Genomics

Plesiomonas shigelloides is the only species in its genus, and has zoonotic importance due to the serious implications resulting from the consumption of contaminated seafood. This is the first report on the genomic features of the whole-genome sequence (WGS) of P. shigelloides strain V-78, recovered from diseased rainbow trout, Oncorhynchus mykiss. The genome of P. shigelloides V-78 consists of 4,478,098 base pairs (bp), which encode 3730 proteins, and has a G + C content of 51.1%. The bioinformatics analysis of WGS of V-78 confirmed the presence of 121 tRNA genes and 42 rRNA genes (15 genes for 5S rRNA, 13 genes for 16S rRNA, and 14 genes for 23S rRNA). Comprehensive genome analyses revealed that the strain encodes for secondary metabolites, antimicrobial resistance, and virulence genes. The strain V-78 has 31 known antibiotic resistance models, which encode many antimicrobial resistances. In addition, strain V-78 has 42 different virulence genes, such as adhesion, secretion system, and motility. The digital DNA–DNA hybridization value against P. shigelloides NCTC 10360 was 74.2%, while the average nucleotide identity value was 97.1%. Based on the scrutinized analysis of genomic data, strain V-78 should be considered a novel subspecies of P. shigelloides, for which Plesiomonas shigelloides subsp. oncorhynchi is proposed.

Dual RNA-Seq of Flavobacterium psychrophilum and Its Outer Membrane Vesicles Distinguishes Genes Associated with Susceptibility to Bacterial Cold-Water Disease in Rainbow Trout (Oncorhynchus mykiss).

Flavobacterium psychrophilum (Fp), the causative agent of Bacterial Cold-Water disease in salmonids, causes substantial losses in aquaculture. Bacterial outer membrane vesicles (OMVs) contain several virulence factors, enzymes, toxins, and nucleic acids and are expected to play an essential role in host-pathogen interactions. In this study, we used transcriptome sequencing, RNA-seq, to investigate the expression abundance of the protein-coding genes in the Fp OMVs versus the Fp whole cell. RNA-seq identified 2190 transcripts expressed in the whole cell and 2046 transcripts in OMVs. Of them, 168 transcripts were uniquely identified in OMVs, 312 transcripts were expressed only in the whole cell, and 1878 transcripts were shared in the two sets. Functional annotation analysis of the OMV-abundant transcripts showed an association with the bacterial translation machinery and histone-like DNA-binding proteins. RNA-Seq of the pathogen transcriptome on day 5 post-infection of Fp-resistant versus Fp-susceptible rainbow trout genetic lines revealed differential gene expression of OMV-enriched genes, suggesting a role for the OMVs in shaping the host-microbe interaction. Interestingly, a cell wall-associated hydrolase (CWH) gene was the most highly expressed gene in OMVs and among the top upregulated transcripts in susceptible fish. The CWH sequence was conserved in 51 different strains of Fp. The study provides insights into the potential role of OMVs in host-pathogen interactions and explores microbial genes essential for virulence and pathogenesis.