Enteric Redmouth Research Articles

Control of yersiniosis in rainbow trout, Oncorhynchus mykiss: innovative non-antibiotic feed-based strategies

Abstract Yersiniosis or enteric redmouth (ERM) is responsible for significant mortalities among rainbow trout and other salmonids. Hence, its prevention and/or control has become a research hotspot. Antibiotics are effective for control; however, accumulating evidence indicates that unrestricted use of antibiotics harms fish, human beings, and the environment. Thus, finding safe, efficacious, cost-effective, and environmentally friendly substitutes is of great interest. Reports showed that dietary supplementation with functional additives with health-promoting and immunostimulatory functions could be a promising strategy. Herein, we provide an overview of the non-antibiotic feed-based strategies for combating EMR in rainbow trout without resorting to antibiotic usage. For this issue, the uses and application of some vitamins, probiotics, prebiotics, synbiotics, quorum quenching probiotic strains, algae, or organic acids, are included in the article. The roles of phytobiotics (herbal supplements), their phytochemicals, and the essential oils extracted from them will also be elucidated. Some emerging nano-based feed supplements are also discussed. Considering their advantages, literature declared that the dietary supplementation of these supplements could enhance the overall health condition, haemato-immunity, and disease resistance and conferred high protection of trout experimentally infected with yersiniosis. Given this perspective, this article presents the current situation and the latest knowledge on these supplements, emphasizing not only their roles to support immunity and decrease the effects of EMR but also to minimize antibiotic usage in aquaculture. Limitations and future perspectives on their effectiveness and efficacy are also discussed. This review will help enhance aquaculture sustainability and improve the health and welfare of rainbow trout.

Biomarkers of oxidative stress, biochemical changes, and the activity of lysosomal enzymes in the livers of rainbow trout (Oncorhynchus mykiss Walbaum) vaccinated against yersiniosis before a Yersinia ruckeri challenge

Abstract Introduction This study aimed to evaluate biomarkers of oxidative stress (2-thiobarbituric acid reactive substances, aldehyde and ketone derivatives of oxidatively modified proteins and total antioxidant capacity), the activity of antioxidant enzymes (superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase), that of lysosomal enzymes (alanyl aminopeptidase, leucyl aminopeptidase, β-N-acetylglucosaminidase and acid phosphatase) and changes in biochemical parameters (alanine aminotransferase, aspartate aminotransferase, de Ritis ratio, lactate dehydrogenase activity, lactate and pyruvate levels and their ratio) in the liver tissue of fish that were vaccinated against enteric redmouth disease and challenged with its causative agent, the bacterium Yersinia ruckeri. Material and Methods The vaccine was administered orally to trout, some of which were challenged with Y. ruckeri 61 days later. For comparison, unvaccinated and unchallenged trout and unvaccinated and challenged trout were also evaluated. Results In the unvaccinated fish, infection with Y. ruckeri disrupted the pro-oxidant/antioxidant balance, led to a significant increase in lipid peroxidation and oxidative modification of proteins, decreased total antioxidant capacity and significantly increased the activity of lysosomal enzymes. In vaccinated fish, the Y. ruckeri challenge increased the activity of glutathione-related enzymes and decreased lipid peroxidation, anaerobic metabolism and the activity of lysosomal enzymes in fish livers relative to the unvaccinated and challenged group. In contrast, these parameters increased after the Y. ruckeri challenge in unvaccinated trout relative to those in the untreated group. Conclusion Vaccination exerted a protective effect during the Y. ruckeri challenge and had no adverse effect on fish livers.

The Putative Virulence Plasmid pYR4 of the Fish Pathogen Yersinia ruckeri Is Conjugative and Stabilized by a HigBA Toxin-Antitoxin System.

The bacterium Yersinia ruckeri causes enteric redmouth disease in salmonids and hence has substantial economic implications for the farmed fish industry. The Norwegian Y. ruckeri outbreak isolate NVH_3758 carries a relatively uncharacterized plasmid, pYR4, which encodes both type 4 pili and a type 4 secretion system. In this study, we demonstrate that pYR4 does not impose a growth burden on the Y. ruckeri host bacterium, nor does the plasmid contribute to twitching motility (an indicator of type 4 pilus function) or virulence in a Galleria mellonella larval model of infection. However, we show that pYR4 is conjugative. We also reveal, through mutagenesis, that pYR4 encodes a functional post-segregational killing system, HigBA, that is responsible for plasmid maintenance within Y. ruckeri. This is the first toxin-antitoxin system to be characterized for this organism. Whilst further work is needed to elucidate the virulence role of pYR4 and whether it contributes to bacterial disease under non-laboratory conditions, our results suggest that the plasmid possesses substantial stability and transfer mechanisms that imply importance within the organism. These results add to our understanding of the mobile genetic elements and evolutionary trajectory of Y. ruckeri as an important commercial pathogen, with consequences for human food production.

Yersinia ruckeri Infection and Enteric Redmouth Disease among Endangered Chinese Sturgeons, China, 2022.

During October 2022, enteric redmouth disease (ERM) affected Chinese sturgeons at a farm in Hubei, China, causing mass mortality. Affected fish exhibited characteristic red mouth and intestinal inflammation. Investigation led to isolation of a prominent bacterial strain, zhx1, from the internal organs and intestines of affected fish. Artificial infection experiments confirmed the role of zhx1 as the pathogen responsible for the deaths. The primary pathologic manifestations consisted of degeneration, necrosis, and inflammatory reactions, resulting in multiple organ dysfunction and death. Whole-genome sequencing of the bacteria identified zhx1 as Yersinia ruckeri, which possesses 135 drug-resistance genes and 443 virulence factor-related genes. Drug-susceptibility testing of zhx1 demonstrated high sensitivity to chloramphenicol and florfenicol but varying degrees of resistance to 18 other antimicrobial drugs. Identifying the pathogenic bacteria associated with ERM in Chinese sturgeons establishes a theoretical foundation for the effective prevention and control of this disease.

Mitochondrial damage and activation of NLRP3 induced by Yersinia ruckeri infection play an important role in the occurrence of diffuse acute inflammation of channel catfish (Ictalurus punctatus)

Yersinia ruckeri, as a facultative intracellular pathogen, can cause two different infection phenotypes of fish. An important phenotype is diffuse acute inflammation (DAI), which is mainly characterized by systemic hemorrhage and multiple tissues damage. Another phenotype is enteric redmouth symptom (EM) with predominantly digestive system damage, while slight damage to other tissues and organs. At present, the causes and mechanisms of heterogeneity of Y. ruckeri infection are not clear. According to our previous research and related bioinformatics analysis, it is suggested that the heterogeneity of infection of Y. ruckeri may be related to the activation of intracellular pattern recognition receptor (NLRP3). Furthermore, combined with the transmission electron microscope observation and ultrastructural pathological analysis of DAI and EM phenotypes, it is hypothesized that Y. ruckeri infection may mediate the occurrence of DAI by inducing mitochondrial damage and activation of NLRP3. In this study, we established DAI and EM phenotypes of channel catfish (Ictalurus punctatus) through the artificial infection experiment. Subsequently, we observed the pathological changes and characteristics of different tissues in DAI and EM phenotypes, such as liver, spleen, kidney, intestine and gill. In addition, the mitochondrial damage of head kidney mediated by Y. ruckeri infection was confirmed by transmission electron microscope observation. Furthermore, by detecting the expression of proinflammatory factors and related pattern recognition receptors in various tissues, we preliminarily clarified the molecular characteristics of immune response in DAI and EM phenotype fish. Moreover, the head kidney macrophages isolated from DAI and EM were observed through transmission electron microscope, and the expression levels of NLRP3, Caspase-1 and IL-1β were detected. Finally, the correlation between mitochondrial damage induced by Y. ruckeri infection and NLRP3 signal transduction was preliminary revealed. Taken together, our results showed that the inflammatory DAI phenotype of fish mediated by Y. ruckeri infection is closely related to mitochondrial damage and NLRP3 activation, which will provide theoretical basis for further study on the pathogenic mechanism of Y. ruckeri infection and clinical diagnosis of the disease, and also provide some insights into the role of NLRP3 in the natural immunity of aquatic animals.

Tarragon (Artemisia dracunculus) Essential Oil at Optimized Dietary Levels Prompted Growth, Immunity, and Resistance to Enteric Red-Mouth Disease in the Rainbow Trout (Oncorhynchus mykiss)

Fingerlings of the rainbow trout, Oncorhynchus mykiss (n = 300, 10.63 ± 0.6 g), were fed tarragon (Artemisia dracunculus) essential oil (TGO) for 2 months to examine its effects on growth properties, immunity, and resistance to Yersinia ruckeri infection. The treatments were control or TG1, TG2 (fed 0.5% TGO), TG3 (1% TGO), and TG4 (2% TGO). According to the results, an improvement was observed in growth parameters in all TGO-treated groups compared to the control (P<0.05). The digestive enzyme activities (protease and lipase) were significantly elevated in response to dietary TGO (P<0.05). The immune system of the fish was enhanced by TGO, as it stimulated the immune parameters in serum (lysozyme, myeloperoxidase (MPO), alternative complement (ACH50), Ig) and mucus (lysozyme, protease, ACH50, Ig) (P<0.05). The treatments, TG3 and TG4, showed more immune performance in response to TGO (P<0.05). The fish in TG2 treatment had a higher levels of serum total protein than other groups (P<0.05). The concentration of triglycerides (TRIG) and cholesterol (CHOL) in serum significantly decreased (P<0.05) in response to TGO, as the lowest levels were observed in the treatment, TG3. The antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)) of serum elevated in TGO-treated fish, with the maximum values for the TG4 group (P<0.05). TGO reduced (P<0.05) alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels in serum. After bacterial challenge, the TGO-treated fish showed lower mortality compared to the control, where the lowest mortality was observed in TG4 (P<0.05). In conclusion, TGO improved growth, immunity, and survival after bacterial challenge in the rainbow trout, with more performance in fish fed 1%–2% TGO.

Detection of Nucleic Acids of the Fish Pathogen Yersinia ruckeri from Planktonic and Biofilm Samples with a CRISPR/Cas13a-Based Assay.

Yersinia ruckeri is the cause of hemorrhagic septicemia, known as enteric redmouth disease, in salmonid fish species. This bacterial pathogen can form biofilms on abiotic surfaces of aquaculture settings or even on the surfaces of the fish themselves, contributing to their persistence in the aquatic environment. Detection methods for this and other fish pathogens can be time-consuming and lack specificity and sensitivity, limiting timely monitoring, the treatment of microbial infections, and effective control of their transmission in aquaculture settings. Rapid and sensitive detection methods for nucleic acids can be crucial for an appropriate surveillance of bacterial pathogens, and the CRISPR/Cas-based assays have emerged as a good alternative since it has been proven to be a useful tool for the rapid, specific, and sensitive detection of viruses and some bacteria. In this study, we explored the capability of the CRISPR/Cas13a system (SHERLOCK) to specifically detect both DNA and RNA (gene transcripts) from planktonic and biofilm samples of the bacterial fish pathogen Y. ruckeri. The assay was designed to detect the gyrA gene and the small noncoding RNAs (sRNAs) MicA and RprA from planktonic cultures and biofilm samples prepared in marine broth. The specific crRNA designed for these gene targets included a 28 nt specific gene sequence, and a scaffold sequence necessary for Cas13-binding. For all the assays, the nucleic acids obtained from samples were previously subjected to isothermal amplification with the recombinase polymerase amplification (RPA) method and the subsequent T7 transcription of the RPA amplicons. Finally, the detection of nucleic acids of Y. ruckeri was by means of a reporter signal released by the Cas13a collateral RNA cleavage triggered upon target recognition, measured by fluorescence- or lateral-flow-based readouts. This CRISPR/Cas13a-based assay was able to specifically detect both DNA and sRNAs from the Y. ruckeri samples, and the sensitivity was comparable to that obtained with qPCR analysis, highlighting the potential applicability of this CRISPR/Cas13a-based assay for fish pathogen surveillance.

ORAL VACCINATION AGAINST YERSINIA RUCKERI: BIOMARKERS OF PROTEIN OXIDATION IN THE HEPATIC TISSUE OF RAINBOW TROUT (ONCORHYNCHUS MYKISS WALBAUM)

The aim of this study was to evaluate the one-month effect of oral vaccination against Yersinia ruckeri based on oxidative stress biomarkers in the hepatic tissue of rainbow trout (Oncorhynchus mykiss Walbaum). The vaccine consisted of three strains of Y. ruckeri (serotype O1) obtained from rainbow trout from different farms where fish showed clinical signs of enteric redmouth disease. The vaccine was produced at the Department of Fish Diseases, National Veterinary Research Institute in Pulawy (Poland) according to the process covered by patent no. P.428259. The concentrated vaccine was added to the fish feed. Treatment was administered three times at one-day intervals. Livers were sampled one month after immunisation. Our results show that immunisation with the anti-Yersinia vaccine did not alter the liver tissue of rainbow trout. aldehydic and ketonic derivatives of oxidatively modified proteins (OMP) were not significantly altered in the hepatic tissue of vaccinated fish prior to immunisation. One month after immunisation, the levels of ketonic derivatives of OMP in the hepatic tissue of untreated trout were reduced compared to the untreated group before immunisation. Similar to the untreated groups, the levels of ketonic derivatives of OMP in the hepatic tissue of the vaccinated group one month after immunisation were reduced compared to the vaccinated group before immunisation. A statistically non-significant decrease in the total antioxidant capacity (TAC) levels was observed between the values obtained in the untreated and vaccinated groups before vaccination and one month after vaccination. Understanding the role of oxidative stress in the tissues of vaccinated trout has important implications for understanding the complex physiological changes that occur during vaccination, and also for improving aquaculture practices to maximise tissue growth and health of vaccinated trout.

Recombinant expression of Yersinia ruckeri outer membrane proteins in Escherichia coli extracellular vesicles

The secretion of extracellular vesicles (EVs) is a common process in Gram-negative bacteria and can be exploited for biotechnological applications. EVs pose a self-adjuvanting, non-replicative vaccine platform, where membrane and antigens are presented to the host immune system in a non-infectious fashion. The secreted quantity of EVs varies between Gram-negative bacterial species and is comparatively high in the model bacterium E. coli. The outer membrane proteins OmpA and OmpF of the fish pathogen Y. ruckeri have been proposed as vaccine candidates to prevent enteric redmouth disease in aquaculture. In this work, Y.ruckeri OmpA or OmpF were expressed in E. coli and recombinant EVs were isolated. To avoid competition between endogenous E. coli OmpA or OmpF, Y. ruckeri OmpA and OmpF were expressed in E. coli strains lacking ompA, ompF, and in a quadruple knockout strain where the four major outer membrane protein genes ompA, ompC, ompF and lamB were removed. Y.ruckeri OmpA and OmpF were successfully expressed in EVs derived from the E. coli mutants as verified by SDS-PAGE, heat modifiability and proteomic analysis using mass-spectrometry. Transmission electron microscopy revealed the presence of EVs in all E. coli strains, and increased EV concentrations were detected when expressing Y. ruckeri OmpA or OmpF in recombinant EVs compared to empty vector controls as verified by nanoparticle tracking analysis. These results show that E. coli can be utilized as a vector for production of EVs expressing outer membrane antigens from Y. ruckeri.

Yersiniomics, a Multi-Omics Interactive Database for Yersinia Species.

The genus Yersinia includes a large variety of nonpathogenic and life-threatening pathogenic bacteria, which cause a broad spectrum of diseases in humans and animals, such as plague, enteritis, Far East scarlet-like fever (FESLF), and enteric redmouth disease. Like most clinically relevant microorganisms, Yersinia spp. are currently subjected to intense multi-omics investigations whose numbers have increased extensively in recent years, generating massive amounts of data useful for diagnostic and therapeutic developments. The lack of a simple and centralized way to exploit these data led us to design Yersiniomics, a web-based platform allowing straightforward analysis of Yersinia omics data. Yersiniomics contains a curated multi-omics database at its core, gathering 200 genomic, 317 transcriptomic, and 62 proteomic data sets for Yersinia species. It integrates genomic, transcriptomic, and proteomic browsers, a genome viewer, and a heatmap viewer to navigate within genomes and experimental conditions. For streamlined access to structural and functional properties, it directly links each gene to GenBank, the Kyoto Encyclopedia of Genes and Genomes (KEGG), UniProt, InterPro, IntAct, and the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and each experiment to Gene Expression Omnibus (GEO), the European Nucleotide Archive (ENA), or the Proteomics Identifications Database (PRIDE). Yersiniomics provides a powerful tool for microbiologists to assist with investigations ranging from specific gene studies to systems biology studies. IMPORTANCE The expanding genus Yersinia is composed of multiple nonpathogenic species and a few pathogenic species, including the deadly etiologic agent of plague, Yersinia pestis. In 2 decades, the number of genomic, transcriptomic, and proteomic studies on Yersinia grew massively, delivering a wealth of data. We developed Yersiniomics, an interactive web-based platform, to centralize and analyze omics data sets on Yersinia species. The platform allows user-friendly navigation between genomic data, expression data, and experimental conditions. Yersiniomics will be a valuable tool to microbiologists.

Exploring Yersinia ruckeri (O1 Biotype 2) infection in three early life-stages of rainbow trout.

Enteric redmouth disease (ERM) caused by the enterobacterium Yersinia ruckeri poses a significant threat to salmonid aquaculture globally. Despite decades of experimental infection studies, key knowledge gaps remain regarding the onset of disease susceptibility and mechanisms of immunity during early developmental stages, undermining disease management efforts in all susceptible life-stages. In this study, a series of immersion challenges were conducted, challenging and re-challenging rainbow trout Oncorhynchus mykiss (Walbaum) at 7, 14 and 51 d post-hatch (dph; mean weights = 0.085, 0.1 and 2.0 g respectively) to high concentrations (1.72 × 107-1.1 × 108 CFU) of Y. ruckeri at 15°C. This study indicates the hitherto unknown initial point of susceptibility to infection as the time of first ingestion of exogenous food (14 dph), and shows that individuals surviving primary challenge at 14 dph are significantly more likely to survive re-challenge at 51 dph compared with naive individuals (hazard ratio = 1.446, p = 0.032). Other key findings include large variation in mortality between different development-stages, from 21.1% at 14 dph to 81.2% at 51 dph, and novel age-dependent symptoms not reported previously. Results from this study enhance our understanding of ERM in juvenile rainbow trout and inform the development of improved aquatic animal health management strategies, thereby contributing to the productivity and sustainability of salmonid aquaculture into the future.

Inhibitory Effect of Select Nitrocompounds and Chlorate against Yersinia ruckeri and Yersinia aleksiciae In Vitro.

Yersinia ruckeri is an important fish pathogen causing enteric redmouth disease. Antibiotics have traditionally been used to control this pathogen, but concerns of antibiotic resistance have created a need for alternative interventions. Presently, chlorate and certain nitrocompounds were tested against Y. ruckeri as well as a related species within the genus, Y. aleksiciae, to assess the effects of these inhibitors. The results reveal that 9 mM chlorate had no inhibitory effect against Y. ruckeri, but inhibited growth rates and maximum optical densities of Y. aleksciciae by 20-25% from those of untreated controls (0.46 h-1 and 0.29 maximum optical density, respectively). The results further reveal that 2-nitropropanol and 2-nitroethanol (9 mM) eliminated the growth of both Y. ruckeri and Y. aleksiciae during anaerobic or aerobic culture. Nitroethane, ethyl nitroacetate and ethyl-2-nitropropionate (9 mM) were less inhibitory when tested similarly. Results from a mixed culture of Y. ruckeri with fish tank microbes and of Y. aleksiciae with porcine fecal microbes reveal that the anti-Yersinia activity of the tested nitrocompounds was bactericidal, with 2-nitropropanol and 2-nitroethanol being more potent than the other tested nitrocompounds. The anti-Yersinia activity observed with these tested compounds warrants further study to elucidate the mechanisms of action and strategies for their practical application.

Comparative analysis of sturgeon- and catfish-derived Yersinia ruckeri reveals the genetic variation and the risk of heavy antibiotic resistance

Sturgeon (Acipenseriformes) and catfish (Siluriformes) are two of the most famous special economic animals, accounting for 1.3 million tons of production in China. Enteric redmouth disease caused by Yersinia ruckeri is common in these two fishes. This study carried out a surveillance of ERM from April 2016 to August 2018 in the Sichuan province. The genotyping and the distribution of antibiotic resistance profiles (ARPs) and genes (ARGs) of Y. ruckeri were established in this study. The results showed that Y. ruckeri was a common pathogen in catfish and sturgeon, accounting for 25.56 % and 17.65 %, respectively. Sixteen strains were obtained and were clustered into 5 genotypes which has a fish-specific characteristic. Y. ruckeri had appeared resistance to 14 of the 20 antibiotics, and 39 antibiotic resistance genes were detected positively from 95 specific primers. Also, there were significant differences in antibiotic resistance profiles among different strains. The sturgeon-derived Y. ruckeri had higher detection rates of aac(6)-Ib(aka aacA4)-1, aac(6)-Ib(aka aacA4)-2, aac(6)-Ib(aka aacA4)-3, fox5 and acrF, and higher resistance rates to ampicillin, ciprofloxacin, levofloxacin, kanamycin, and neomycin compared with the catfish-derived strains. Our results indicated that Y. ruckeri might become an important pathogen in catfish and sturgeon culture in Sichuan, and there were different subspecies of variation. Besides, Y. ruckeri has enriched many antibiotic resistances that may help evolve into super bacteria.

Neurosteroid Role in the Integration of Olfactory Immune Responses in Rainbow Trout (Oncorhynchus Mykiss)

The objective of this study is to unravel the integration of olfactory signals involved in the nasal detection of pathogens and the activation of associated physiological responses in Rainbow trout.The infectious pathogen Yersinia ruckeri causes enteric redmouth disease in aquaculture farms worldwide. Recent research shows that olfactory organ of rainbow trout is highly susceptible external tissue like gills or skin due to direct contact with the microbiome‐rich environment. Recent findings have demonstrated that olfactory sensory cells play a key role in early pathogen detection and immune cell activation along with their primary role in detecting environmental stimuli.We hypothesize that olfactory immune responses elicited by Y ruckeri are modulated by neurosteroids. To test our hypothesis, we nasally exposed rainbow trout to Y. ruckeri vaccine or saline and took nose and brain samples at four different time periods (4 hours, 24 hours, 7 days, and 14 days) after exposure. We analyzed fish neurosteroids (cortisol, estradiol, and progesterone) and gene expression of steroidogenic enzymes (p450, 3β HSD, 11β HSD, and 17 β HSD) in nose, olfactory bulb, and rest of the brain. Exposure to Y. rukeri vaccine caused significant changes in tissue neurosteroid production and steroidogenic enzyme expression at different times after exposure.Within the olfactory epithelium (OE), we detected a significant decrease (p = 0.003) in estradiol after 15 min exposure with 26.1 pg/mg tissue and 391.0 pg/mg tissue in treated and control individual respectively. Progesterone (174.83 pg/mg tissue) was significantly (p = 0.004) higher after 4 hours compared to the control (26.5 pg/mg of tissue) and cortisol (10.95 pg/mg of tissue) was significantly lower (p = 0.003) after 14 days in Y. ruckeri‐treated fish compared to the control (78.76 pg/mg of tissue). Within the olfactory bulb (OB), lower concentrations of cortisol and progesterone (5.65 pg/mg tissue and 1.6 pg/mg tissue respectively) were observed after 15 minutes in the Y. ruckeri‐treated fish compared to the control (22.8 pg/mg of tissue and 1.9 pg/mg of tissue, respectively). There was also a higher concentration of estradiol after 15 minutes and 4 hours compared to the control. Lastly, within the rest of brain cortisol (9.1 pg/mg tissue) was significantly higher after 4 hours in vaccine treated fish compared to the control (4.9 pg/mg tissue) and estradiol was significantly lower after 15 minutes in the vaccine group (5.5 pg/mg of tissue), compared to the control (55.9 pg/mg of tissue). However, neurosteroid concentration in the rest of brain was relatively low compared to the OE and OB. The neurosteroids: cortisol, estradiol, and progesterone play many important roles in fish physiology including stress response, cognitive behavioral modulation, and immunological response. Our preliminary results also show a significant differential expression of steroidogenic enzymes after exposure to the vaccine. Altogether, our results demonstrate that neurosteroids play a critical role in the integration of immune olfactory responses in vertebrates.

Integrative analyses of probiotics, pathogenic infections and host immune response highlight the importance of gut microbiota in understanding disease recovery in rainbow trout (Oncorhynchus mykiss).

Given the pivotal role played by the gut microbiota in regulating the host immune system, great interest has arisen in the possibility of controlling fish health by modulating the gut microbiota. Hence, there is a need to better understand of the host-microbiota interactions after disease responses to optimize the use of probiotics to strengthen disease resilience and recovery. We tested the effects of a probiotic feed additive in rainbow trout and challenged the fish with the causative agent for enteric red mouth disease, Yersinia ruckeri. We evaluated the survival, host immune gene expression and the gut microbiota composition. Results revealed that provision of probiotics and exposure to Y. ruckeri induced immune gene expression in the host, which were associated with changes in the gut microbiota. Subsequently, infection with Y. ruckeri had very little effect on microbiota composition when probiotics were applied, indicating that probiotics increased stabilisation of the microbiota. Our analysis revealed potential biomarkers for monitoring infection status and fish health. Finally, we used modelling approaches to decipher interactions between gut bacteria and the host immune gene responses, indicating removal of endogenous bacteria elicited by non-specific immune responses. We discuss the relevance of these results emphasizing the importance of host-microbiota interactions, including the protective potential of the gut microbiota in disease responses. Our results highlight the functional consequences of probiotic-induced changes in the gut microbiota post infection and the resulting host immune response.

Functional and Molecular Immune Response of Rainbow Trout (Oncorhynchus mykiss) Following Challenge with Yersinia ruckeri

Currently, aquaculture production of rainbow trout (Oncorhynchus mykiss) is a multibillion dollar industry; nevertheless, the development of this sector has not been exempt from pitfalls related to the recurrent presence of pathogens of bacterial origin. This is the case of Yersinia ruckeri, the etiologic agent of the infectious pathology known as Enteric Red Mouth Disease (ERM), causing serious economic losses that can be as high as 30–70% of production. Although several studies have been performed regarding pathogen features and virulence factors, more information is needed about the host defense mechanism activation after infection. Given this perspective, this study aimed to evaluate rainbow trout’s short-term innate immune response against infection with Y. ruckeri. A series of factors linked to the innate immune response were evaluated, including determination of hematological parameters, oxidative stress biomarkers, and analysis of the expression of immune-related genes. Results showed a significant decrease in several hematological parameters (white blood cell count, hematocrit, neutrophils, monocytes, lymphocytes, and thrombocytes) and oxidative stress indicators (SOD) between the control and infected groups. In addition, there were significant differences in the level of gene expression between infected individuals and the control group. Most of these genes (il-1β, il-8, il-10, tnf-α1, tnf-α2, socs3, mmp-9, cath, hsp-70, saa, fer, pcb) were upregulated within the first 24 h following infection. Results from this study showed more insights into the short-term immune response of rainbow trout to infection with Y. ruckeri, which may be useful for the establishment of biomarkers that may be used for the early detection of ERM.

Heterogeneity of Yersinia ruckeri Isolated from Rainbow Trout (Oncorhynchus mykiss) Infected with Enteric Red Mouth Disease

Regarding the increasing prevalence of antimicrobial resistance as a global menace, typing procedures are of great importance in epidemiological surveys. In the current study, interspecies differences of Yersinia ruckeri (Y. ruckeri) isolates were detected by antimicrobial susceptibility profiling and molecular traits to determine the relationship between isolates. The current study was conducted on 27 Y. ruckeri isolates collected from 40 infected rainbow trout (Oncorhynchus mykiss) with clinical enteric red mouth disease in Mashhad, Iran. Interspecies differences of all isolates were detected by antimicrobial susceptibility profiling using disk diffusion method, Repetitive Extragenic Palindromic PCR (REP-PCR), Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), and Random Amplification of Polymorphic DNA PCR (RAPD-PCR) to determine the relationship between isolates. Simpson’s diversity index was calculated for each typing technique. The most phenotype resistant was against ampicillin, chloramphenicol, and lincomycin. Furthermore, enrofloxacin and ciprofloxacin showed the highest activity (100%) against isolates. According to the results, the highest diversity index was observed in the REP-PCR method (D = 0.91). In conclusion, REP-PCR can be a powerful technique for epidemiological studies.

ORAL VACCINATION AGAINST YERSINIA RUCKERI: OXIDATIVE STRESS BIOMARKERS IN THE GILLS OF RAINBOW TROUT (ONCORHYNCHUS MYKISS WALBAUM)

The aim of this study was to assess the effect of oral vaccination against Yersinia ruckeri based on oxidative stress biomarkers in the gills of rainbow trout (Oncorhynchus mykiss Walbaum). The vaccine consisted of three Y. ruckeri strains (O1 serotype) that originated from rainbow trout cultured on different farms, where fish exhibited clinical signs of enteric redmouth disease. The concentrated vaccine was incorporated into the fish food; treatment was delivered three times at one-day intervals. Two months after immunization, gills were sampled. The vaccination against Y. ruckeri resulted in non-significant changes in TBARS level as lipid peroxidation marker, aldehydic and ketonic derivatives of oxidatively modified proteins level in the gills of trout at the second month after immunization against Y. ruckeri. A significant decrease in superoxide dismutase activity (by 36%, p = 0.002) compared to untreated controls occurred. The alterations in markers of oxidative stress suggest that antioxidant defenses may contribute to the balance between oxidative modification of proteins and antioxidant defenses in the gills of trout vaccinated against Y. ruckeri. We did not find any alterations in the gills after 60 days of immunization. This is likely a result of long-term adaptation to immunization. Understanding the role of oxidative stress in the tissues of vaccinated trout has important implications for the understanding of the complex physiological changes that occur in immunization and also for improving aquaculture practices to maximize tissue growth and health of vaccinated trout. The oxidative stress biomarkers in the gills were sensitive to vaccination against Y. ruckeri and may potentially be used as biomarkers in evaluating vaccine toxicity in rainbow trout. From a practical point of view, the results may be useful in relation to studies of infections and the development, administration, and uptake of new vaccines applicable to large amounts of fish.

Ruckerbactin Produced by Yersinia ruckeri YRB Is a Diastereomer of the Siderophore Trivanchrobactin Produced by Vibrio campbellii DS40M4.

The Gram-negative bacterium Yersinia ruckeri is the causative agent for enteric red mouth disease in salmonids. The genome of Y. ruckeri YRB contains a biosynthetic gene cluster encoding the biosynthesis of catechol siderophores that are diastereomeric with the known vanchrobactin class of siderophores, (DHBDArgLSer)(1-3). Ruckerbactin (1), produced by Y. ruckeri YRB, was found to be the linear tris-l-serine ester composed of l-arginine and 2,3-dihydroxybenzoic acid, (DHBLArgLSer)3. The biscatechol, (DHBLArgLSer)2 (2), and monocatechol, DHBLArgLSer (3), compounds were also isolated and characterized. The macrolactone of ruckerbactin was not detected. The presence of LArg in ruckerbactin makes it the diastereomer of trivanchrobactin with DArg. The electronic circular dichroism spectra of Fe(III)-ruckerbactin and Fe(III)-trivanchrobactin reveal the opposite enantiomeric configurations at the Fe(III) sites. Fe(III)-ruckerbactin adopts the Δ configuration, and Fe(III)-trivanchrobactin adopts the Λ configuration. Y. ruckeri YRB was also found to produce the antimicrobial agent holomycin (4).

Dual RNA-Seq of Trunk Kidneys Extracted From Channel Catfish Infected With Yersinia ruckeri Reveals Novel Insights Into Host-Pathogen Interactions.

Host-pathogen intectarions are complex, involving large dynamic changes in gene expression through the process of infection. These interactions are essential for understanding anti-infective immunity as well as pathogenesis. In this study, the host-pathogen interaction was analyzed using a model of acute infection where channel catfish were infected with Yersinia ruckeri. The infected fish showed signs of body surface hyperemia as well as hyperemia and swelling in the trunk kidney. Double RNA sequencing was performed on trunk kidneys extracted from infected channel catfish and transcriptome data was compared with data from uninfected trunk kidneys. Results revealed that the host-pathogen interaction was dynamically regulated and that the host-pathogen transcriptome fluctuated during infection. More specifically, these data revealed that the expression levels of immune genes involved in Cytokine-cytokine receptor interactions, the NF-kappa B signaling pathway, the JAK-STAT signaling pathway, Toll-like receptor signaling and other immune-related pathways were significantly upregulated. Y. ruckeri mainly promote pathogenesis through the flagellum gene fliC in channel catfish. The weighted gene co-expression network analysis (WGCNA) R package was used to reveal that the infection of catfish is closely related to metabolic pathways. This study contributes to the understanding of the host-pathogen interaction between channel catfish and Y. ruckeri, more specifically how catfish respond to infection through a transcriptional perspective and how this infection leads to enteric red mouth disease (ERM) in these fish.