Tyrosine-modifying glycosylation by Yersinia effectors.

Mono-O-glycosylation of target proteins by bacterial toxins or effector proteins is a well-known mechanism by which bacteria interfere with essential functions of host cells. The respective glycosyltransferases are important virulence factors such as the Clostridioides difficile toxins A and B. Here, we describe two glycosyltransferases of Yersinia species that have a high sequence identity: YeGT from the zoonotic pathogen Yersinia enterocolitica and YkGT from the murine pathogen Yersinia kristensenii. We show that both modify Rho family proteins by attachment of GlcNAc at tyrosine residues (Tyr-34 in RhoA). Notably, the enzymes differed in their target protein specificity. While YeGT modified RhoA, B, and C, YkGT possessed a broader substrate spectrum and glycosylated not only Rho but also Rac and Cdc42 subfamily proteins. Mutagenesis studies indicated that residue 177 is important for this broader target spectrum. We determined the crystal structure of YeGT shortened by 16 residues N terminally (sYeGT) in the ligand-free state and bound to UDP, the product of substrate hydrolysis. The structure assigns sYeGT to the GT-A family. It shares high structural similarity to glycosyltransferase domains from toxins. We also demonstrated that the 16 most N-terminal residues of YeGT and YkGT are important for the mediated translocation into the host cell using the pore-forming protective antigen of anthrax toxin. Mediated introduction into HeLa cells or ectopic expression of YeGT and YkGT caused morphological changes and redistribution of the actin cytoskeleton. The data suggest that YeGT and YkGT are likely bacterial effectors belonging to the family of tyrosine glycosylating bacterial glycosyltransferases.

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.

Epidemiological cut-off values for Yersina ruckeri disc diffusion data generated by a standardised method.

In order to establish the meaning of data generated in antimicrobial agent susceptibility tests, it is necessary to develop internationally harmonised interpretive criteria. Currently, such criteria have not been developed for data generated in studies of the susceptibility of the fish pathogen Yersinia ruckeri. This work generated the data that would be required to set epidemiological cut-off values for the susceptibility data of this species that had been generated using a standardised disc diffusion method that specified the use of Mueller Hinton agar and incubation at 22°C for 24-28 h. Using this method, sets of inhibition zones data for 4 antimicrobial agents were generated by 3 independent laboratories. The data from these laboratories were aggregated and analysed using the statistically based normalised resistance interpretation. For ampicillin, florfenicol, oxytetracycline and trimethoprim-sulfamethoxazole the cut-off values calculated by this analysis were ≥16, ≥23, ≥24 and ≥30 mm, respectively. Evidence is presented demonstrating that the data for these 4 agents was of sufficient quantity and quality that they could be used by the relevant authorities to set internationally harmonised, consensus epidemiological cut-off values for Y. ruckeri.

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.

Mechanism of sturgeon intestinal inflammation induced by Yersinia ruckeri and the effect of florfenicol intervention.

The mechanism by which Y. ruckeri infection induces enteritis in Chinese sturgeon remains unclear, and the efficacy of drug prevention and control measures is not only poor but also plagued with numerous issues. We conducted transcriptomic and 16 S rRNA sequencing analyses to examine the differences in the intestinal tract of hybrid sturgeon before and after Y. ruckeri infection and florfenicol intervention. Our findings revealed that Y. ruckeri induced the expression of multiple inflammatory factors, including il1ß, il6, and various chemokines, as well as casp3, casp8, and multiple tumor necrosis factor family members, resulting in pathological injury to the body. Additionally, at the phylum level, the relative abundance of Firmicutes and Bacteroidota increased, while the abundance of Plesiomonas and Cetobacterium decreased at the genus level, altering the composition of the intestinal flora. Following florfenicol intervention, the expression of multiple apoptosis and inflammation-related genes was down-regulated, promoting tissue repair. However, the flora became further dysregulated, increasing the risk of infection. In conclusion, our analysis of the transcriptome and intestinal microbial composition demonstrated that Y. ruckeri induces intestinal pathological damage by triggering apoptosis and altering the composition of the intestinal microbiota. Florfenicol intervention can repair pathological damage, but it also exacerbates flora imbalance, leading to a higher risk of infection. These findings help elucidate the molecular mechanism of Y. ruckeri-induced enteritis in sturgeon and evaluate the therapeutic effect of drugs on intestinal inflammation in sturgeon.

Cell wall disruption, membrane damage, and decrease in the expression of Yrp1 virulence factor in Yersinia ruckeri by propolis ethanol extract.

Background and Objectives: Instead of antibiotics, propolis is a promising alternative for treating bacterial diseases. The aim of this study was to evaluate the effect of propolis ethanol extract (PEE) on Yersinia ruckeri (Y. ruckeri), a fish pathogen, by examining its impact on the cell wall, cytoplasmic membrane, and gene expression. Materials and Methods: The effect of propolis on the bacterial cell wall, membrane, and DNA using scanning electron microscopy (SEM) was investigated. Its effect on the NAD+/NADH ratio, reactive oxygen species (ROS) production, as well as the expression of a virulence factor (yrp1) was also determined. Results: It was demonstrated that PEE has multiple antibacterial mechanisms against Y. ruckeri involving cell wall damage, membrane lysis, and a decrease in gene expression. Conclusion: The obtained results indicated that the mode of propolis action against Y. ruckeri is both structural and functional, while others showed propolis only could inactivate bacteria in a structural way.

New Evidence for the Role of Pituitary Adenylate Cyclase-Activating Polypeptide as an Antimicrobial Peptide in Teleost Fish.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is a multifunctional neuropeptide that is widely distributed and conserved across species. We have previously shown that in teleost fish, PACAP not only possesses direct antimicrobial properties but also immunomodulatory effects against the bacterial pathogens Flavobacterium psychrophilum and Pseudomonas aeruginosa using in vitro and in vivo experiments. These previous results suggest PACAP can be used as an alternative to antibiotics to prevent and/or treat bacterial infections in the aquaculture industry. To accomplish this goal, more studies are needed to better understand the effect of PACAP on pathogens affecting fish in live infections. In the present study, the transcripts PACAP, PRP/PACAP, and VPAC2 receptor were examined in rainbow trout (Oncorhynchus mykiss) naturally infected with Yersinia ruckeri, which exhibited an increase in their expression in the spleen when compared to healthy fish. Synthetic Clarias gariepinus PACAP-38 has direct antimicrobial activity on Y. ruckeri and inhibits up to 60% of the bacterial growth when the peptide is at concentrations between 50 and 100 µM in TSB. The growth inhibition increased up to 90% in the presence of 12.5 µM of PACAP-38 when salt-free LB broth was used instead of TSB. It was also found to inhibit Y. ruckeri growth in a dose-dependent manner when the rainbow trout monocyte/macrophage-like cell line (RTS11) was pre-treated with lower concentrations of the peptide (0.02 and 0.1 µM) before going through infection. Differential gene expression was analyzed in this in vitro model. Overall, the results revealed new evidence to support the role of PACAP as an antimicrobial and immunomodulatory peptide treatment in teleosts.

In vitro modelling of the influence of alternative feeds (Hermetia illucens, Arthrospira platensis) on the resistance of different rainbow trout populations (Oncorhynchus mykiss) against the viral haemorrhagic septicaemia virus and Yersinia ruckeri.

Replacing fishmeal, a finite resource with high market demand, in the diet of carnivorous rainbow trout with proteins from alternative sources may be a challenge for these fish. Therefore, this study investigated whether replacing fishmeal with protein derived from Hermetia illucens or Arthrospira platensis could promote disease susceptibility in local trout populations with different growth performance. This was assessed in vitro by measuring susceptibility to infection with the viral haemorrhagic septicaemia virus (VHSV) or the bacterium Yersinia ruckeri. Analysis of fin tissue explants and primary cell cultures from scales from the three trout populations infected in vitro with VHSV and gill explants infected with Y. ruckeri showed no significant differences in virus replication or bacterial counts. Evaluation of the virucidal or bactericidal effect of skin mucus showed a significant reduction in viral load and bacterial count for all samples with mucus addition, but no significant difference was observed between the experimental groups. This study documents no apparent impairment of innate immune mechanisms in the skin and gills of trout after feeding a diet replacing fishmeal with Arthrospira or Hermetia proteins. This underlines the potential of these alternative protein sources for the further development of sustainable trout aquaculture.

Effects of diets rich in Agaricus bisporus polysaccharides on the growth, antioxidant, immunity, and resistance to Yersinia ruckeri in channel catfish.

To promote the application of Agaricus bisporus polysaccharides (ABPs) in channel catfish (Ictalurus punctatus) culture, we evaluated the effects of ABPs on the growth, immunity, antioxidant, and antibacterial activity of channel catfish. When the amount of ABPs was 250 mg/kg, channel catfish's weight gain and specific growth rates increased significantly while the feed coefficient decreased. We also found that adding ABPs in the feed effectively increased the activities of ACP, MDA, T-SOD, AKP, T-AOC, GSH, and CAT enzymes and immune-related genes such as IL-1ß, Hsp70, and IgM in the head kidney of channel catfish. Besides, long-term addition will not cause pathological damage to the head kidney. When the amount of ABPs was over 125 mg/kg, the protection rate of channel catfish was more than 60%. According to the intestinal transcriptome analysis, the addition of ABPs promoted the expression of intestinal immunity genes and growth metabolism-related genes and enriched multiple related KEEG pathways. When challenged by Yersinia ruckeri infection, the immune response of channel catfish fed with ABPs was intenser and quicker. Additionally, the 16S rRNA gene sequencing analysis showed that the composition of the intestinal microbial community of channel catfish treated with ABPs significantly changed, and the abundance of microorganisms beneficial to channel catfish growth, such as Firmicutes and Bacteroidota increased. In conclusion, feeding channel catfish with ABPs promoted growth, enhanced immunity and antioxidant, and improved resistance to bacterial infections. Our current results might promote the use of ABPs in channel catfish and even other aquacultured fish species.

Screening and activity of potential gastrointestinal probiotic lactic acid bacteria against Yersinia ruckeri O1b.

Yersiniosis of cultured Atlantic salmon is a recurrent fish health management challenge in many continents. The causative organism, Yersinia ruckeri, can reside latently in the gut and lead to acute infection and disease during hatchery and sea-transfer stages. One potential prevention approach is the administration of probiotic bacteria to suppress gut colonization of Y. ruckeri. Our study aimed to isolate and identify anti-Yersinia activity among lactic acid bacteria (LAB) isolated from the gastrointestinal tract (GIT) of aquatic animals. Of the 186 aquatic GIT isolates examined, three strains showed diffusible antimicrobial activity towards Y. ruckeri O1b. Analysis of 16 s rRNA gene sequences indicated the three bacterial strains were Enterococci, related to Enterococcus sp. (99%), Enterococcus thailandicus (99%), and Enterococcus durans (99%). Anti-Yersinia activity was maintained at neutral pH (~6.5-7.0), and in-vitro environmental tolerance assays showed the three strains could withstand simulated salmonids gastrointestinal tract conditions of: low pH (3.4) and 3% bile salt content. All three Enterococci strains showed higher adhesion to the intestinal mucus of Atlantic salmon than Y. ruckeri O1b (E. durans 24%, E. enterococcus sp. 25% and E. thailandicus 98%, compared to Y. ruckeri O1b 5%). However, only Enterococcus sp. and E. thailandicus were able to grow in the salmon intestinal mucus broth while E. durans showed no growth. Anti-Yersinia activity was completely inactivated by proteinase-K treatment, suggesting that the active compound/s are proteinaceous and may be bacteriocin-like inhibitory substances (BLIS). Our data indicate that Enterococcus sp. MA176 and E. thailandicus MA122 are potential probionts for the prevention of yersiniosis in salmonids. Further in-vivo studies are required to determine whether these bacteria reduce the incidence of yersiniosis in Atlantic salmon.

First preliminary study on identification of bacterial fish pathogens with Raman spectroscopy.

Accurate and rapid determination of bacterial disease agents of fish is an important step for sustainable and efficient aquaculture production. In general, biochemical and molecular methods are used for pathogen detection but they are usually time-consuming and required qualified personnel. Recently spectroscopic methods are preferred in clinical and food microbiology and declared as a promising alternative method for pathogens diagnosis with many advantages. In this study, the significant spectra of three important bacterial fish pathogens (Lactococcus garvieae, Vibrio anguillarum and Yersinia ruckeri) were determined by Raman spectroscopy. The first data of the pathogens were obtained and the distinctive differences in polysaccharides, nucleic acids, fatty acids and amino acids were identified. This preliminary study aimed to be pioneer for further studies in aquaculture and veterinary microbiology toward developing an alternative method for routine identification.

Development and validation of main spectral profile for rapid identification of Yersinia ruckeri isolated from Atlantic salmon using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows rapid and reliable identification of microorganisms. The accuracy of bacterial identification using MALDI-TOF MS depends on main spectral profiles (MSPs) provided in a quality-assured commercial reference library, which requires ongoing improvement. This study aimed to develop and validate an in-house MALDI-TOF MS MSP to rapidly identify Yersinia ruckeri isolated from Atlantic salmon (Salmo salar). The novel MSP was prepared using an isolate of Y. ruckeri recovered from Atlantic salmon and confirmed by 16S rRNA gene sequencing. Subsequently, a validation set which comprises 29 isolates of Y. ruckeri were examined from three fishes: Atlantic salmon (Salmo salar) (n = 26), American eel (Anguilla rostrata) (n = 1), and Atlantic cod (Gadus morhua) (n = 2). These isolates were randomly selected from the Atlantic Veterinary College, Aquatic Diagnostic Services Bacteriology Laboratory's culture collection to validate the novel MSP. Analytical sensitivity of MALDI-TOF MS using the novel MSP to identify the validation set was 86.2%. Repeatability was assessed by acquiring spectra from 30 different spots of a randomly-selected isolate of Y. ruckeri, and analyzed spectra from each spot were compared against the novel MSP. The coefficient of variation was 3.3%. The novel MSP clustered with Bruker MSPs (n = 3) of Y. ruckeri in the reference library and did not falsely identify any closely related bacteria to Y. ruckeri. This study reports the development of a novel MSP of high analytical sensitivity and specificity for rapid identification of Y. ruckeri using MALDI-TOF MS.

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.

Corrigendum: Dual RNA-seq of trunk kidneys extracted from channel catfish infected with Yersinia ruckeri reveals novel insights into host-pathogen interactions.

[This corrects the article DOI: 10.3389/fimmu.2021.775708.].

Therapeutic Intervention with Dietary Chitosan Nanoparticles Alleviates Fish Pathological and Molecular Systemic Inflammatory Responses against Infections.

Marine bio-sourced chitosan nanoparticles (CSNP) are antimicrobial and immunomodulatory agents beneficial for fish medicine. Herein, dietary CSNP was investigated for the amelioration of the systemic inflammatory responses of an induced fish model. One hundred and forty-four rainbow trout were assigned to one pathogen-free and non-supplemented group (negative control), and three challenged groups: non-supplemented (positive control), CSNP-preventive, and CSNP-therapeutic. After a feeding experiment extended for 21 days, the organosomatic indices (OSI) and molecular aspects were assessed. After a challenge experiment extended for further 28 days, CSNP-therapeutic intervention was assessed on fish survival and systemic inflammatory responses on pathology, histo-morphology, and molecular aspects. With CSNP administration, OSI nonsignificantly decreased and the relative expression of targeted inflammatory-mediator genes was significantly increased. The CSNP-therapeutic fish showed an RPS of 80% as compared to the positive control group, and CSNP-therapeutic administration retained the highest gene expression augmentation up to 28 days after the challenge. Notably, the splenic reticulin fibers framework of the CSNP-therapeutic group retained the highest integrity among the groups during the infection. After recovery, reticulin fibers density in the CSNP-therapeutic samples was significantly higher than in the negative control group, which indicates high innate immunity. Thus, CSNP showed promising biotherapeutic features enhancing fish resistance against infections.

Kinetics of the invasion of a non-phagocytic fish cell line, RTG-2 by Yersinia ruckeri serotype O1 biotype 1.

Yersiniosis, caused by the fish pathogen Yersinia ruckeri, is a serious bacterial septicaemia affecting mainly salmonids worldwide. The acute infection may result in high mortality without apparent external disease signs, while the chronic one causes moderate to considerable mortality. Survivors of yersiniosis outbreaks become carriers. Y. ruckeri is able to adhere to, and to invade, phagocytic and non-phagocytic fish cells by using unknown molecular mechanisms. The aim of this study was to describe the kinetics of cell invasion by Y. ruckeri serotype O1 biotype 1 in a fish cell line (RTG-2) originating from rainbow trout gonads. The efficiency of invasion by Y. ruckeri was found to be temperature dependent, having a maximum at 20 °C. The bacterium was able to survive up to 96 h postinfection. The incubation of the cells at 4 °C and the pre-incubation of the bacteria with sugars or heat-inactivated antiserum significantly decreased the efficiency of invasion or even completely prevented the invasion of RTG-2 cells. These findings indicate that Y. ruckeri is capable of adhering to, entering and surviving within non-phagocytic cells, and that the intracellular environment may constitute a suitable niche for this pathogen that can favour the spread of infection and/or the maintenance of a carrier state of fish.

qPCR screening for Yersinia ruckeri clonal complex 1 against a background of putatively avirulent strains in Norwegian aquaculture.

Although a number of genetically diverse Yersinia ruckeri strains are present in Norwegian aquaculture environments, most if not all outbreaks of yersiniosis in Atlantic salmon in Norway are associated with a single specific genetic lineage of serotype O1, termed clonal complex 1. To investigate the presence and spread of virulent and putatively avirulent strains in Norwegian salmon farms, PCR assays specific for Y. ruckeri (species level) and Y. ruckeri clonal complex 1 were developed. Following extensive screening of water and biofilm, the widespread prevalence of putatively avirulent Y. ruckeri strains was confirmed in freshwater salmon hatcheries, while Y. ruckeri clonal complex 1 was found in fewer farms. The formalin-killed bacterin yersiniosis vaccine was detected in environmental samples by both PCR assays for several weeks post-vaccination. It is thus important to interpret results from recently vaccinated fish with great care. Moreover, field studies and laboratory trials confirmed that stressful management procedures may result in increased shedding of Y. ruckeri by sub-clinically infected fish. Analysis of sea water sampled throughout thermal delousing procedures proved effective for detection of Y. ruckeri in sub-clinically infected populations.

Stress response and virulence factors in bacterial pathogens relevant for Chilean aquaculture: current status and outlook of our knowledge.

The study of the stress responses in bacteria has given us a wealth of information regarding the mechanisms employed by these bacteria in aggressive or even non-optimal living conditions. This information has been applied by several researchers to identify molecular targets related to pathogeny, virulence, and survival, among others, and to design new prophylactic or therapeutic strategies against them. In this study, our knowledge of these mechanisms has been summarized with emphasis on some aquatic pathogenic bacteria of relevance to the health and productive aspects of Chilean salmon farming (Piscirickettsia salmonis, Tenacibaculum spp., Renibacterium salmoninarum, and Yersinia ruckeri). This study will aid further investigations aimed at shedding more light on possible lines of action for these pathogens in the coming years.

Biotyping reveals loss of motility in two distinct Yersinia ruckeri lineages exclusive to Norwegian aquaculture.

Non-motile strains of Yersinia ruckeri, known as Y. ruckeri biotype 2, now dominate amongst clinical isolates retrieved from rainbow trout internationally. Due to an acute increase in the number of yersiniosis cases in Norway in recent years, followed by introduction of widespread intraperitoneal vaccination against the disease, an investigation on the prevalence of Y. ruckeri biotype 2 in Norwegian aquaculture was conducted. We biotyped 263 Y. ruckeri isolates recovered from diseased salmonids in Norway between 1985 and 2020. A total of seven biotype 2 isolates were identified, four of which were collected between 1985 and 1987, and three of which belong to the current epizootic clone, isolated from two different sea-farms in 2017. Whole-genome sequencing revealed single non-synonymous nucleotide polymorphisms in the flagellar genes flhC in isolates from the 1980s, and in fliP in isolates from 2017. In both variants, motility was restored both by complementation with wild-type alleles in trans and via spontaneous mutation-driven reversion following prolonged incubation on motility agar. While biotype 2 strains do not yet seem to have become broadly established in Norwegian aquaculture, the seven isolates described here serve to document a further two independent cases of Y. ruckeri biotype 2 emergence in salmonid aquaculture.

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).

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results highlight the functional consequences of probiotic-induced changes in the gut microbiota post infection and the resulting host immune response.